Modulators of alpha-synuclein toxicity

ABSTRACT

Disclosed are genes that, when overexpressed in cells expressing alpha-synuclein, either suppress or enhance alpha-synuclein mediated cellular toxicity. Compounds that modulate expression of these genes or activity of the encoded proteins can be used to inhibit alpha-synuclein mediated toxicity and used to treat or prevent synucleinopathies such as Parkinson&#39;s disease. Also disclosed are methods of identifying inhibitors of alpha-synuclein mediated toxicity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application No.61/016,214, filed Dec. 21, 2007, and U.S. Provisional Application No.61/090,797, filed Aug. 21, 2008. The entire content of each of theseprior applications is incorporated herein by reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under grant number 2P50NS038372-0681 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

TECHNICAL FIELD

This invention relates to compositions and methods for inhibitingalpha-synuclein mediated toxicity and methods for identifying inhibitorsof alpha-synuclein mediated toxicity.

BACKGROUND

Parkinson's disease is a neurodegenerative disorder that ispathologically characterized by the presence of intracytoplasmic Lewybodies (Lewy in Handbuch der Neurologie, M. Lewandowski, ed., Springer,Berlin, pp. 920-933, 1912; Pollanen et al., J. Neuropath. Exp. Neurol.52:183-191, 1993), the major components of which are filamentsconsisting of alpha-synuclein (Spillantini et al., Proc. Natl. Acad.Sci. USA 95:6469-6473, 1998; Arai et al., Neurosci. Lett. 259:83-86,1999), a 140-amino acid protein (Ueda et al., Proc. Natl. Acad. Sci. USA90:11282-11286, 1993). Two dominant mutations in alpha-synuclein causingfamilial early onset Parkinson's disease have been described, suggestingthat Lewy bodies contribute mechanistically to the degeneration ofneurons in Parkinson's disease and related disorders (Polymeropoulos etal., Science 276:2045-2047, 1997; Kruger et al., Nature Genet.18:106-108, 1998; Zarranz et al., Ann. Neurol. 55:164-173, 2004).Triplication and duplication mutation of the alpha-synuclein gene havebeen linked to early-onset of Parkinson's disease (Singleton et al.,Science 302:841, 2003; Chartier-Harlin at al. Lancet 364:1167-1169,2004; Ibanez et al., Lancet 364:1169-1171, 2004). In vitro studies havedemonstrated that recombinant alpha-synuclein can indeed form Lewybody-like fibrils (Conway et al., Nature Med. 4:1318-1320, 1998;Hashimoto et al., Brain Res. 799:301-306, 1998; Nahri et al., J. Biol.Chem. 274:9843-9846, 1999). Both Parkinson's disease-linkedalpha-synuclein mutations accelerate this aggregation process,demonstrating that such in vitro studies may have relevance forParkinson's disease pathogenesis. Alpha-synuclein aggregation and fibrilformation fulfills the criteria of a nucleation-dependent polymerizationprocess (Wood et al., J. Biol. Chem. 274:19509-19512, 1999). In thisregard alpha-synuclein fibril formation resembles that of Alzheimer'sβ-amyloid protein (Aβ) fibrils. Alpha-synuclein recombinant protein, andnon-Aβ component (known as NAC), which is a 35-amino acid peptidefragment of alpha-synuclein, both have the ability to form fibrils whenincubated at 37° C., and are positive with amyloid stains such as Congored (demonstrating a red/green birefringence when viewed under polarizedlight) and Thioflavin S (demonstrating positive fluorescence) (Hashimotoet al., Brain Res. 799:301-306, 1998; Ueda et al., Proc. Natl. Acad.Sci. USA 90:11282-11286, 1993).

Synucleins are a family of small, presynaptic neuronal proteins composedof α-, β-, and γ-synucleins, of which only alpha-synuclein aggregateshave been associated with several neurological diseases (Ian et al.,Clinical Neurosc. Res. 1:445-455, 2001; Trojanowski and Lee,Neurotoxicology 23:457-460, 2002). The role of synucleins (and inparticular, alpha-synuclein) in the etiology of a number ofneurodegenerative and/or amyloid diseases has developed from severalobservations. Pathologically, alpha-synuclein was identified as a majorcomponent of Lewy bodies, the hallmark inclusions of Parkinson'sdisease, and a fragment thereof was isolated from amyloid plaques of adifferent neurological disease, Alzheimer's disease. Biochemically,recombinant alpha-synuclein was shown to form amyloid-like fibrils thatrecapitulated the ultrastructural features of alpha-synuclein isolatedfrom patients with dementia with Lewy bodies, Parkinson's disease andmultiple system atrophy. Additionally, the identification of mutationswithin the alpha-synuclein gene, albeit in rare cases of familialParkinson's disease, demonstrated an unequivocal link between synucleinpathology and neurodegenerative diseases. The common involvement ofalpha-synuclein in a spectrum of diseases such as Parkinson's disease,dementia with Lewy bodies, multiple system atrophy and the Lewy bodyvariant of Alzheimer's disease has led to the classification of thesediseases under the umbrella term of “synucleinopathies.”

Fibrillization and aggregation of alpha-synuclein is thought to playmajor role in neuronal dysfunction and death of dopaminergic neurons inParkinson's disease. Mutations in alpha-synuclein or genomictriplication of wild type alpha-synuclein (leading to itsoverexpression) cause certain rare familial forms of Parkinson'sdisease. In vitro and in vivo models suggest that over-expression ofwild-type alpha-synuclein induces neuronal cell death. See, e.g.,Polymeropoulos, et al. (1997) Science 276(5321):2045-7, Kruger, et al.(1998) Nat Genet. 18(2):106-8, Singleton, et al. (2003) Science302(5646):841, Miller, et al. (2004) Neurology 62(10):1835-8, Hashimoto,et al. (2003) Ann N Y Acad Sci. 991:171-88, Lo Bianco, et al. (2002)Proc Natl Acad Sci U S A. 99(16):10813-8, Lee, et al. (2002) Proc NatlAcad Sci U S A. 99(13):8968-73, Masliah, et al. (2000) Science287(5456):1265-9, Auluck, et al. (2002) Science 295(5556):865-8,Oluwatosin-Chigbu et al. (2003) Biochem Biophys Res Commun 309(3):679-84, Klucken et al. (2004) J Biol Chem. 279(24):25497-502. Protectingneurons from the toxic effects of alpha-synuclein is a promisingstrategy for treating Parkinson's disease and other synucleinopathiessuch as Lewy body dementia.

Thus, there is a need for compounds and compositions that preventalpha-synuclein toxicity and/or aggregation and/or promotealpha-synuclein fibril disaggregation. Such compounds and compositionsare useful in treating or ameliorating one or more symptoms ofalpha-synuclein mediated diseases and disorders, or diseases anddisorders in which alpha-synuclein toxicity is implicated, including butnot limited to, Parkinson's disease (including Parkinson's diseasechemically induced by exposure to environmental agents such aspesticides, insecticides, or herbicides and/or metals such as manganese,aluminum, cadmium, copper, or zinc, SNCA gene-linked Parkinson'sdisease, sporadic or idiopathic Parkinson's disease, or Parkin- orLRRK2-linked Parkinson's disease), dementia with Lewy bodies, pureautonomic failure, multiple system atrophy, incidental Lewy bodydisease, pantothenate kinase-associated neurodegeneration, Alzheimer'sdisease, Down's Syndrome, Gaucher disease, or the Parkinsonism-dementiacomplex of Guam.

SUMMARY

The invention is based, at least in part, on the discovery that certaingenes, when overexpressed in cells expressing alpha-synuclein, eithersuppress or enhance alpha-synuclein mediated cellular toxicity. Theidentification of these genes as relevant to alpha-synuclein mediatedtoxicity permits the carrying out of screens to identify compounds thatmodulate toxicity. Compounds identified by such screens can be used ascandidate drugs for the treatment or prevention of synucleinopathiessuch as Parkinson's disease.

The disclosure features methods of inhibiting alpha synuclein-mediatedcellular toxicity by contacting a cell expressing a toxicity-inducingamount or form of alpha synuclein with an effective amount of a compoundthat inhibits expression or activity of GOS1, SEC31, IZH3, MKS1, TPO4,GOSR1, SEC31A, SEC31B, ADIPOR2, ADIPOR1, NRG1, NRG2, BET4, GLO3, SLY41,TRS120, YIP3, IDS2, PPZ2, UBP11, UBP7, SIP5, MATALPHA1, KLF12, KLFS,ZNF323, ZNF718, ZNF705A, RABGGTA, ZNF289, SLC35E1, NIBP, RABAC1,KIAA0999, PPP1CC, PPP1CB, PPP1CA, USP21, or USP2.

Also disclosed are methods of inhibiting alpha synuclein-mediatedcellular toxicity by contacting a cell expressing a toxicity-inducingamount or form of alpha synuclein with an effective amount of a compoundthat inhibits expression or activity of NRG1, NRG2, BET4, GLO3, SLY41,TRS120, YIP3, IDS2, PPZ2, UBP11, UBP7, SIP5, MATALPHA1, KLF12, KLFS,ZNF323, ZNF718, ZNF705A, RABGGTA, ZNF289, SLC35E1, NIBP, RABAC1,KIAA0999, PPP 1CC, PPP1CB, PPP1CA, USP21, or USP2.

In some embodiments, the compound used in the methods contains a nucleicacid that inhibits translation of an RNA encoding the protein. In otherembodiments, the compound used in the methods contains a nucleic acidthat inhibits transcription of a DNA encoding the protein. Alsodisclosed are methods of identifying a compound that inhibits alphasynuclein-mediated toxicity by: (i) providing a cell expressing anamount or form of alpha synuclein that reduces viability of the cell;(ii) contacting the cell with an agent that inhibits expression oractivity of GOS1, SEC31, IZH3, MKS1, TPO4, GOSR1, SEC31A, SEC31B,ADIPOR2, ADIPOR1, NRG1, NRG2, BET4, GLO3, SLY41, TRS120, YIP3, IDS2,PPZ2, UBP11, UBP7, SIP5, MATALPHA1, KLF12, KLF5, ZNF323, ZNF718,ZNF705A, RABGGTA, ZNF289, SLC35E1, NIBP, RABAC1, KIAA0999, PPP1CC,PPP1CB, PPP1CA, USP21, or USP2; and (iii) measuring cell viability inthe presence of the agent, wherein an increase in cell viability in thepresence of the agent as compared to cell viability in the absence ofthe agent identifies the agent as a compound that inhibits alphasynuclein-mediated toxicity.

Also disclosed are methods of identifying a compound that inhibits alphasynuclein-mediated toxicity by: (i) providing a cell expressing anamount or form of alpha synuclein that reduces viability of the cell;(ii) contacting the cell with an agent that inhibits expression oractivity of NRG1, NRG2, BET4, GLO3, SLY41, TRS120, YIP3, IDS2, PPZ2,UBP11, UBP7, SIP5, MATALPHA1, KLF12, KLF5, ZNF323, ZNF718, ZNF705A,RABGGTA, ZNF289, SLC35E1, NIBP, RABAC1, KIAA0999, PPP1CC, PPP1CB,PPP1CA, USP21, or USP2; and (iii) measuring cell viability in thepresence of the agent, wherein an increase in cell viability in thepresence of the agent as compared to cell viability in the absence ofthe agent identifies the agent as a compound that inhibits alphasynuclein-mediated toxicity.

Also disclosed are methods of identifying a compound that inhibits alphasynuclein-mediated toxicity by: (i) screening to identify an agent thatinhibits expression or activity of GOS1, SEC31, IZH3, MKS1, TPO4, GOSR1,SEC31A, SEC31B, ADIPOR2, ADIPOR1, NRG1, NRG2, BET4, GLO3, SLY41, TRS120,YIP3, IDS2, PPZ2, UBP11, UBP7, SIP5, MATALPHA1, KLF12, KLF5, ZNF323,ZNF718, ZNF705A, RABGGTA, ZNF289, SLC35E1, NIBP, RABAC1, KIAA0999,PPP1CC, PPP1CB, PPP1CA, USP21, or USP2; (ii) providing a cell expressingan amount or form of alpha synuclein that reduces viability of the cell;(iii) contacting the cell with the agent; and (iv) measuring cellviability in the presence of the agent, wherein an increase in cellviability in the presence of the agent as compared to cell viability inthe absence of the agent identifies the agent as a compound thatinhibits alpha synuclein-mediated toxicity.

Also disclosed are methods of identifying a compound that inhibits alphasynuclein-mediated toxicity by: (i) screening to identify an agent thatinhibits expression or activity of NRG1, NRG2, BET4, GLO3, SLY41,TRS120, YIP3, IDS2, PPZ2, UBP11, UBP7, SIP5, MATALPHA1, KLF12, KLF5,ZNF323, ZNF718, ZNF705A, RABGGTA, ZNF289, SLC35E1, NIBP, RABAC1,KIAA0999, PPP1CC, PPP1CB, PPP1CA, USP21, or USP2; (ii) providing a cellexpressing an amount or form of alpha synuclein that reduces viabilityof the cell; (iii) contacting the cell with the agent; and (iv)measuring cell viability in the presence of the agent, wherein anincrease in cell viability in the presence of the agent as compared tocell viability in the absence of the agent identifies the agent as acompound that inhibits alpha synuclein-mediated toxicity.

Also disclosed are methods of identifying a compound that inhibitsexpression of a protein by: (i) providing a cell expressing GOS1, SEC31,IZH3, MKS1, TPO4, GOSR1, SEC31A, SEC31B, ADIPOR2, ADIPOR1, NRG1, NRG2,BET4, GLO3, SLY41, TRS120, YIP3, IDS2, PPZ2, UBP11, UBP7, SIP5,MATALPHA1, KLF12, KLF5, ZNF323, ZNF718, ZNF705A, RABGGTA, ZNF289,SLC35E1, NIBP, RABAC1, KIAA0999, PPP1CC, PPP1CB, PPP1CA, USP21, or USP2;(ii) contacting the cell with an agent; and (iii) measuring theexpression of the protein in the presence of the agent, wherein areduction in the expression of the protein in the presence of the agentas compared to the expression of the protein in the absence of the agentidentifies the agent as a compound that inhibits the expression of theprotein.

Also disclosed are methods of identifying a compound that inhibitsexpression of a protein by: (i) providing a cell expressing NRG1, NRG2,BET4, GLO3, SLY41, TRS120, YIP3, IDS2, PPZ2, UBP11, UBP7, SIP5,MATALPHA1, KLF12, KLF5, ZNF323, ZNF718, ZNF705A, RABGGTA, ZNF289,SLC35E1, NIBP, RABAC1, KIAA0999, PPP1CC, PPP1CB, PPP1CA, USP21, or USP2;(ii) contacting the cell with an agent; and (iii) measuring theexpression of the protein in the presence of the agent, wherein areduction in the expression of the protein in the presence of the agentas compared to the expression of the protein in the absence of the agentidentifies the agent as a compound that inhibits the expression of theprotein.

Also disclosed are methods of identifying a compound that inhibitsexpression of a protein by: (i) providing a cell containing a reporterconstruct containing (a) a promoter sequence of a gene encoding GOS1,SEC31, IZH3, MKS1, TPO4, GOSR1, SEC31A, SEC31B, ADIPOR2, ADIPOR1, NRG1,NRG2, BET4, GLO3, SLY41, TRS120, YIP3, IDS2, PPZ2, UBP11, UBP7, SIP5,MATALPHA1, KLF12, KLF5, ZNF323, ZNF718, ZNF705A, RABGGTA, ZNF289,SLC35E1, NIBP, RABAC1, KIAA0999, PPP1CC, PPP1CB, PPP1CA, USP21, or USP2,and (b) a nucleotide sequence encoding a reporter protein; (ii)contacting the cell with an agent; and (iii) measuring the expression ofthe reporter protein in the presence of the agent, wherein a reductionin the expression of the reporter protein in the presence of the agentas compared to the expression of the reporter protein in the absence ofthe agent identifies the agent as a compound that inhibits theexpression of the protein.

Also disclosed are methods of identifying a compound that inhibitsexpression of a protein by: (i) providing a cell containing a reporterconstruct containing (a) a promoter sequence of a gene encoding NRG1,NRG2, BET4, GLO3, SLY41, TRS120, YIP3, IDS2, PPZ2, UBP11, UBP7, SIP5,MATALPHA1, KLF12, KLF5, ZNF323, ZNF718, ZNF705A, RABGGTA, ZNF289,SLC35E1, NIBP, RABAC1, KIAA0999, PPP1CC, PPP1CB, PPP1CA, USP21, or USP2,and (b) a nucleotide sequence encoding a reporter protein; (ii)contacting the cell with an agent; and (iii) measuring the expression ofthe reporter protein in the presence of the agent, wherein a reductionin the expression of the reporter protein in the presence of the agentas compared to the expression of the reporter protein in the absence ofthe agent identifies the agent as a compound that inhibits theexpression of the protein.

Also disclosed are methods of identifying a compound that inhibits theactivity of a protein by: (i) providing a GOS1, SEC31, IZH3, MKS1, TPO4,GOSR1, SEC31A, SEC31B, ADIPOR2, ADIPOR1, NRG1, NRG2, BET4, GLO3, SLY41,TRS120, YIP3, IDS2, PPZ2, UBP11, UBP7, SIP5, MATALPHA1, KLF12, KLF5,ZNF323, ZNF718, ZNF705A, RABGGTA, ZNF289, SLC35E1, NIBP, RABAC1,KIAA0999, PPP1CC, PPP1CB, PPP1CA, USP21, or USP2 protein; (ii)contacting the protein with an agent; and (iii) measuring the activityof the protein in the presence of the agent, wherein a reduction in theactivity of the protein in the presence of the agent as compared to theactivity of the protein in the absence of the agent identifies the agentas a compound that inhibits the activity the protein.

Also disclosed are methods of identifying a compound that inhibits theactivity of a protein by: (i) providing a NRG1, NRG2, BET4, GLO3, SLY41,TRS120, YIP3, IDS2, PPZ2, UBP11, UBP7, SIP5, MATALPHA1, KLF12, KLF5,ZNF323, ZNF718, ZNF705A, RABGGTA, ZNF289, SLC35E1, NIBP, RABAC1,KIAA0999, PPP1CC, PPP1CB, PPP1CA, USP21, or USP2 protein; (ii)contacting the protein with an agent; and (iii) measuring the activityof the protein in the presence of the agent, wherein a reduction in theactivity of the protein in the presence of the agent as compared to theactivity of the protein in the absence of the agent identifies the agentas a compound that inhibits the activity the protein.

In the foregoing methods, the agent can be a synthetic compound or anaturally occurring compound. For example, the agent can be a smallmolecule, nucleic acid, protein, antibody, or peptidomimetic. The cellused in the methods can be a eukaryotic cell (e.g., a yeast cell,nematode cell, insect cell, or mammalian cell), a prokaryotic cell, or acell obtained from an alpha-synuclein transgenic animal.

Also disclosed are methods of treating or preventing a synucleinopathyby administering to a subject in need thereof a pharmaceuticalcomposition containing a therapeutic or prophylactic amount of acompound that decreases the expression or activity of GOSR1, SEC31A,SEC31B, ADIPOR2, ADIPOR1, KLF12, KLF5, ZNF323, ZNF718, ZNF705A, RABGGTA,ZNF289, SLC35E1, NIBP, RABAC1, KIAA0999, PPP1CC, PPP1CB, PPP1CA, USP21,and/or USP2.

Also disclosed are methods of treating or preventing a synucleinopathyby administering to a subject in need thereof a pharmaceuticalcomposition containing a therapeutic or prophylactic amount of acompound that decreases the expression or activity of KLF12, KLF5,ZNF323, ZNF718, ZNF705A, RABGGTA, ZNF289, SLC35E1, NIBP, RABAC1,KIAA0999, PPP1CC, PPP1CB, PPP1CA, USP21, and/or USP2.

Also disclosed are methods of treating or preventing a synucleinopathyby administering to a subject in need thereof a pharmaceuticalcomposition containing a therapeutic or prophylactic amount of acompound that enhances the expression or activity of SLC7A2, SLC7A13,SLC7A6, GBL, DSSP, UGP2, COPG, COPG2, COPE, BET1, ICK, MAK, ULK3, MARK2,MARK1, MARK3, MARK4, ULK1, CAMK1G, CAMK1, CAMK1D, CSNK1G3, CSNK1G2,CSNK1G1, CSNK1D1, CSNK1E1, CSNK1A1, CSNK1A1L, LSG1, OSBPL1A, ERC2,OSBPL6, OSBPL3, OSBPL7, OSBPL1A, OSBPL2, CFTF2T, CFTF2, EEF1G, PPM1G,PPM1A, PPM1B, TREH, PKNOX2, PKNOX1, PUM1, PUM2, EIF4G3, EIF4G1, SYVN1,ZNF364, SETD6, PPCDC, EGR3, ZFP161, EGR2, HKR1, ZNF740, ATP13A2 (PARK9),ATP13A1, ATP13A3, ATP13A4, or ATP13A5.

In some embodiments of the foregoing methods, the synucleinopathy isParkinson's disease (including Parkinson's disease chemically induced byexposure to environmental agents such as pesticides, insecticides, orherbicides and/or metals such as manganese, aluminum, cadmium, copper,or zinc, SNCA gene-linked Parkinson's disease, sporadic or idiopathicParkinson's disease, or Parkin- or LRRK2-linked Parkinson's disease),dementia with Lewy bodies, pure autonomic failure, multiple systematrophy, incidental Lewy body disease, pantothenate kinase-associatedneurodegeneration, Alzheimer's disease, Down's Syndrome, Gaucherdisease, or the Parkinsonism-dementia complex of Guam. A compound thatenhances the expression or activity of a protein includes, for example,a compound that inhibits the degradation of the protein.

In some embodiments of the foregoing methods, the pharmaceuticalcomposition contains (i) a therapeutic or prophylactic amount of SLC7A2,SLC7A13, SLC7A6, GBL, DSSP, UGP2, COPG, COPG2, COPE, BET1, ICK, MAK,ULK3, MARK2, MARK1, MARK3, MARK4, ULK1, CAMK1G, CAMK1, CAMK1D, CSNK1G3,CSNK1G2, CSNK1G1, CSNK1D1, CSNK1E1, CSNK1A1, CSNK1A1L, LSG1, OSBPL1A,ERC2, OSBPL6, OSBPL3, OSBPL7, OSBPL1A, OSBPL2, CFTF2T, CFTF2, EEF1G,PPM1G, PPM1A, PPM1B, TREH, PKNOX2, PKNOX1, PUM1, PUM2, EIF4G3, EIF4G1,SYVN1, ZNF364, SETD6, PPCDC, EGR3, ZFP161, EGR2, HKR1, ZNF740, ATP13A2(PARK9), ATP13A1, ATP13A3, ATP13A4, or ATP13A5, and (ii) apharmaceutically acceptable carrier.

Also disclosed are methods of inhibiting alpha synuclein-mediatedcellular toxicity by contacting a cell expressing a toxicity-inducingamount or form of alpha synuclein with an effective amount of a compoundthat enhances expression or activity of QDR3, DIP5, LST8, REG1, UGP1,SEC21, SEC28, SFT1, IME2, KSP1, RCK2, YCK3, LSG1, MGA2, MUM2, OSH3,PIN4, URE2, PTC4, NTH1, CUP9, JSN1, TIF4632, HRD1, SAN1, YBR030w,YKL088w, YML081w, YMR111c, YOR291W, YOR062C, GI53, PTP2, SKO1, HAP4,VHR1, SUM1, STB3, PFS1, OSH2, ISN1, SLC7A2, SLC7A13, SLC7A6, GBL, DSSP,UGP2, COPG, COPG2, COPE, BET1, ICK, MAK, ULK3, MARK2, MARK1, MARK3,MARK4, ULK1, CAMK1G, CAMK1, CAMK1D, CSNK1G3, CSNK1G2, CSNK1G1, CSNK1D1,CSNK1E1, CSNK1A1, CSNK1A1L, LSG1, OSBPL1A, ERC2, OSBPL6, OSBPL3, OSBPL7,OSBPL1A, OSBPL2, CFTF2T, CFTF2, EEF1G, PPM1G, PPM1A, PPM1B, TREH,PKNOX2, PKNOX1, PUM1, PUM2, EIF4G3, EIF4G1, SYVN1, ZNF364, SETD6, PPCDC,EGR3, ZFP161, EGR2, HKR1, ZNF740, ATP (PARK9), ATP13A1, ATP13A3,ATP13A4, or ATP13A5.

Also disclosed are methods of inhibiting alpha synuclein-mediatedcellular toxicity by contacting a cell expressing a toxicity-inducingamount or form of alpha synuclein with an effective amount of a compoundthat enhances expression or activity of DIP5, LST8, REG1, UGP1, SEC21,SEC28, SFT1, IME2, KSP1, RCK2, YCK3, LSG1, MGA2, MUM2, OSH3, PIN4, URE2,PTC4, NTH1, CUP9, JSN1, TIF4632, HRD1, SAN1, YBR030w, YKL088w, YML081w,YMR111c, YOR291W, YOR062C, GIS3, PTP2, SKO1, HAP4, VHR1, SUM1, STB3,PFS1, OSH2, ISN1, SLC7A2, SLC7A13, SLC7A6, GBL, DSSP, UGP2, COPG, COPG2,COPE, BET1, ICK, MAK, ULK3, MARK2, MARK1, MARK3, MARK4, ULK1, CAMK1G,CAMK1, CAMK1D, CSNK1G3, CSNK1G2, CSNK1G1, CSNK1D1, CSNK1E1, CSNK1A1,CSNK1A1L, LSG1, OSBPL1A, ERC2, OSBPL6, OSBPL3, OSBPL7, OSBPL1A, OSBPL2,CFTF2T, CFTF2, EEF1G, PPM1G, PPM1A, PPM1B, TREH, PKNOX2, PKNOX1, PUM1,PUM2, EIF4G3, EIF4G1, SYVN1, ZNF364, SETD6, PPCDC, EGR3, ZFP161, EGR2,HKR1, ZNF740, ATP (PARK9), ATP13A1, ATP13A3, ATP13A4, or ATP13A5.

Also disclosed are methods of identifying a compound that inhibits alphasynuclein-mediated toxicity by: (i) providing a cell expressing anamount or form of alpha synuclein that reduces viability of the cell;(ii) contacting the cell with an agent that enhances expression oractivity of QDR3, DIP5, LST8, REG1, UGP1, SEC21, SEC28, SFT1, IME2,KSP1, RCK2, YCK3, LSG1, MGA2, MUM2, OSH3, PIN4, URE2, PTC4, NTH1, CUP9,JSN1, TIF4632, HRD1, SAN1, YBR030w, YKL088w, YML081w, YMR111c, YOR291W,YOR062C, GIS3, PTP2, SKO1, HAP4, VHR1, SUM1, STB3, PFS1, OSH2, ISN1,SLC7A2, SLC7A13, SLC7A6, GBL, DSSP, UGP2, COPG, COPG2, COPE, BET1, ICK,MAK, ULK3, MARK2, MARK1, MARK3, MARK4, ULK1, CAMK1G, CAMK1, CAMK1D,CSNK1G3, CSNK1G2, CSNK1G1, CSNK1D1, CSNK1E1, CSNK1A1, CSNK1A1L, LSG1,OSBPL1A, ERC2, OSBPL6, OSBPL3, OSBPL7, OSBPL1A, OSBPL2, CFTF2T, CFTF2,EEF1G, PPM1G, PPM1A, PPM1B, TREH, PKNOX2, PKNOX1, PUM1, PUM2, EIF4G3,EIF4G1, SYVN1, ZNF364, SETD6, PPCDC, EGR3, ZFP161, EGR2, HKR1, ZNF740,ATP13A2 (PARK9), ATP13A1, ATP13A3, ATP13A4, or ATP13A5; and (iii)measuring cell viability in the presence of the agent, wherein anincrease in cell viability in the presence of the agent as compared tocell viability in the absence of the agent identifies the agent as acompound that inhibits alpha synuclein-mediated toxicity.

Also disclosed are methods of identifying a compound that inhibits alphasynuclein-mediated toxicity by: (i) providing a cell expressing anamount or form of alpha synuclein that reduces viability of the cell;(ii) contacting the cell with an agent that enhances expression oractivity of DIP5, LST8, REG1, UGP1, SEC21, SEC28, SFT1, IME2, KSP1,RCK2, YCK3, LSG1, MGA2, MUM2, OSH3, PIN4, URE2, PTC4, NTH1, CUP9, JSN1,TIF4632, HRD1, SAN1, YBR030w, YKL088w, YML081w, YMR111c, YOR291W,YOR062C, GIS3, PTP2, SKO1, HAP4, VHR1, SUM1, STB3, PFS1, OSH2, ISN1,SLC7A2, SLC7A13, SLC7A6, GBL, DSSP, UGP2, COPG, COPG2, COPE, BET1, ICK,MAK, ULK3, MARK2, MARK1, MARK3, MARK4, ULK1, CAMK1G, CAMK1, CAMK1D,CSNK1G3, CSNK1G2, CSNK1G1, CSNK1D1, CSNK1E1, CSNK1A1, CSNK1A1L, LSG1,OSBPL1A, ERC2, OSBPL6, OSBPL3, OSBPL7, OSBPL1A, OSBPL2, CFTF2T, CFTF2,EEF1G, PPM1G, PPM1A, PPM1B, TREH, PKNOX2, PKNOX1, PUM1, PUM2, EIF4G3,EIF4G1, SYVN1, ZNF364, SETD6, PPCDC, EGR3, ZFP161, EGR2, HKR1, ZNF740,ATP13A2 (PARK9), ATP13A1, ATP13A3, ATP13A4, or ATP13A5; and (iii)measuring cell viability in the presence of the agent, wherein anincrease in cell viability in the presence of the agent as compared tocell viability in the absence of the agent identifies the agent as acompound that inhibits alpha synuclein-mediated toxicity.

Also disclosed are methods of identifying a compound that inhibits alphasynuclein-mediated toxicity by: (i) screening to identify an agent thatenhances expression or activity of QDR3, DIP5, LST8, REG1, UGP1, SEC21,SEC28, SFT1, IME2, KSP1, RCK2, YCK3, LSG1, MGA2, MUM2, OSH3, PIN4, URE2,PTC4, NTH1, CUP9, JSN1, TIF4632, HRD1, SAN1, YBR030w, YKL088w, YML081w,YMR111c, YOR291W, YOR062C, GIS3, PTP2, SKO1, HAP4, VHR1, SUM1, STB3,PFS1, OSH2, ISN1, SLC7A2, SLC7A13, SLC7A6, GBL, DSSP, UGP2, COPG, COPG2,COPE, BET1, ICK, MAK, ULK3, MARK2, MARK1, MARK3, MARK4, ULK1, CAMK1G,CAMK1, CAMK1D, CSNK1G3, CSNK1G2, CSNK1G1, CSNK1D1, CSNK1E1, CSNK1A1,CSNK1A1L, LSG1, OSBPL1A, ERC2, OSBPL6, OSBPL3, OSBPL7, OSBPL1A, OSBPL2,CFTF2T, CFTF2, EEF1G, PPM1G, PPM1A, PPM1B, TREH, PKNOX2, PKNOX1, PUM1,PUM2, EIF4G3, EIF4G1, SYVN1, ZNF364, SETD6, PPCDC, EGR3, ZFP161, EGR2,HKR1, ZNF740, ATP13A2 (PARK9), ATP13A1, ATP13A3, ATP13A4, or ATP13A5;(ii) providing a cell expressing an amount or form of alpha synucleinthat reduces viability of the cell; (iii) contacting the cell with theagent; and (iv) measuring cell viability in the presence of the agent,wherein an increase in cell viability in the presence of the agent ascompared to cell viability in the absence of the agent identifies theagent as a compound that inhibits alpha synuclein-mediated toxicity.

Also disclosed are methods of identifying a compound that inhibits alphasynuclein-mediated toxicity by: (i) screening to identify an agent thatenhances expression or activity of DIP5, LST8, REG1, UGP1, SEC21, SEC28,SFT1, IME2, KSP1, RCK2, YCK3, LSG1, MGA2, MUM2, OSH3, PIN4, URE2, PTC4,NTH1, CUP9, JSN1, TIF4632, HRD1, SAN1, YBR030w, YKL088w, YML081w,YMR111c, YOR291W, YOR062C, GIS3, PTP2, SKO1, HAP4, VHR1, SUM1, STB3,PFS1, OSH2, ISN1, SLC7A2, SLC7A13, SLC7A6, GBL, DSSP, UGP2, COPG, COPG2,COPE, BET1, ICK, MAK, ULK3, MARK2, MARK1, MARK3, MARK4, ULK1, CAMK1G,CAMK1, CAMK1D, CSNK1G3, CSNK1G2, CSNK1G1, CSNK1D1, CSNK1E1, CSNK1A1,CSNK1A1L, LSG1, OSBPL1A, ERC2, OSBPL6, OSBPL3, OSBPL7, OSBPL1A, OSBPL2,CFTF2T, CFTF2, EEF1G, PPM1G, PPM1A, PPM1B, TREH, PKNOX2, PKNOX1, PUM1,PUM2, EIF4G3, EIF4G1, SYVN1, ZNF364, SETD6, PPCDC, EGR3, ZFP161, EGR2,HKR1, ZNF740, ATP13A2 (PARK9), ATP13A1, ATP13A3, ATP13A4, or ATP13A5;(ii) providing a cell expressing an amount or form of alpha synucleinthat reduces viability of the cell; (iii) contacting the cell with theagent; and (iv) measuring cell viability in the presence of the agent,wherein an increase in cell viability in the presence of the agent ascompared to cell viability in the absence of the agent identifies theagent as a compound that inhibits alpha synuclein-mediated toxicity.

Also disclosed are methods of identifying a compound that increasesexpression of a protein by: (i) providing a cell expressing a QDR3,DIP5, LST8, REG1, UGP1, SEC21, SEC28, SFT1, IME2, KSP1, RCK2, YCK3,LSG1, MGA2, MUM2, OSH3, PIN4, URE2, PTC4, NTH1, CUP9, JSN1, TIF4632,HRD1, SAN1, YBR030w, YKL088w, YML081w, YMR111c, YOR291W, YOR062C, GIS3,PTP2, SKO1, HAP4, VHR1, SUM1, STB3, PFS1, OSH2, ISN1, SLC7A2, SLC7A13,SLC7A6, GBL, DSSP, UGP2, COPG, COPG2, COPE, BET1, ICK, MAK, ULK3, MARK2,MARK1, MARK3, MARK4, ULK1, CAMK1G, CAMK1, CAMK1D, CSNK1G3, CSNK1G2,CSNK1G1, CSNK1D1, CSNK1E1, CSNK1A1, CSNK1A1L, LSG1, OSBPL1A, ERC2,OSBPL6, OSBPL3, OSBPL7, OSBPL1A, OSBPL2, CFTF2T, CFTF2, EEF1G, PPM1G,PPM1A, PPM1B, TREH, PKNOX2, PKNOX1, PUM1, PUM2, EIF4G3, EIF4G1, SYVN1,ZNF364, SETD6, PPCDC, EGR3, ZFP161, EGR2, HKR1, ZNF740, ATP (PARK9),ATP13A1, ATP13A3, ATP13A4, or ATP protein; (ii) contacting the cell withan agent; and (iii) measuring the expression of the protein in thepresence of the agent, wherein an increase in the expression of theprotein in the presence of the agent as compared to the expression ofthe protein in the absence of the agent identifies the agent as acompound that increases the expression of the protein.

Also disclosed are methods of identifying a compound that increasesexpression of a protein by: (i) providing a cell expressing a DIP5,LST8, REG1, UGP1, SEC21, SEC28, SFT1, IME2, KSP1, RCK2, YCK3, LSG1,MGA2, MUM2, OSH3, PIN4, URE2, PTC4, NTH1, CUP9, JSN1, TIF4632, HRD1,SAN1, YBR030w, YKL088w, YML081w, YMR111c, YOR291W, YOR062C, GIS3, PTP2,SKO1, HAP4, VHR1, SUM1, STB3, PFS1, OSH2, ISN1, SLC7A2, SLC7A13, SLC7A6,GBL, DSSP, UGP2, COPG, COPG2, COPE, BET1, ICK, MAK, ULK3, MARK2, MARK1,MARK3, MARK4, ULK1, CAMK1G, CAMK1, CAMK1D, CSNK1G3, CSNK1G2, CSNK1G1,CSNK1D1, CSNK1E1, CSNK1A1, CSNK1A1L, LSG1, OSBPL1A, ERC2, OSBPL6,OSBPL3, OSBPL7, OSBPL1A, OSBPL2, CFTF2T, CFTF2, EEF1G, PPM1G, PPM1A,PPM1B, TREH, PKNOX2, PKNOX1, PUM1, PUM2, EIF4G3, EIF4G1, SYVN1, ZNF364,SETD6, PPCDC, EGR3, ZFP161, EGR2, HKR1, ZNF740, ATP13A2 (PARK9),ATP13A1, ATP13A3, ATP13A4, or ATP13A5 protein; (ii) contacting the cellwith an agent; and (iii) measuring the expression of the protein in thepresence of the agent, wherein an increase in the expression of theprotein in the presence of the agent as compared to the expression ofthe protein in the absence of the agent identifies the agent as acompound that increases the expression of the protein.

Also disclosed are methods of identifying a compound that increasesexpression of a protein by: (i) providing a cell containing a reporterconstruct containing (a) a promoter sequence of a gene encoding QDR3,DIP5, LST8, REG1, UGP1, SEC21, SEC28, SFT1, IME2, KSP1, RCK2, YCK3,LSG1, MGA2, MUM2, OSH3, PIN4, URE2, PTC4, NTH1, CUP9, JSN1, TIF4632,HRD1, SAN1, YBR030w, YKL088w, YML081w, YMR111c, YOR291W, YOR062C, GIS3,PTP2, SKO1, HAP4, VHR1, SUM1, STB3, PFS1, OSH2, ISN1, SLC7A2, SLC7A13,SLC7A6, GBL, DSSP, UGP2, COPG, COPG2, COPE, BET1, ICK, MAK, ULK3, MARK2,MARK1, MARK3, MARK4, ULK1, CAMK1G, CAMK1, CAMK1D, CSNK1G3, CSNK1G2,CSNK1G1, CSNK1D1, CSNK1E1, CSNK1A1, CSNK1A1L, LSG1, OSBPL1A, ERC2,OSBPL6, OSBPL3, OSBPL7, OSBPL1A, OSBPL2, CFTF2T, CFTF2, EEF1G, PPM1G,PPM1A, PPM1B, TREH, PKNOX2, PKNOX1, PUM1, PUM2, EIF4G3, EIF4G1, SYVN1,ZNF364, SETD6, PPCDC, EGR3, ZFP161, EGR2, HKR1, ZNF740, ATP13A2 (PARK9),ATP13A1, ATP13A3, ATP13A4, or ATP13A5, and (b) a nucleotide sequenceencoding a reporter protein; (ii) contacting the cell with an agent; and(iii) measuring the expression of the reporter protein in the presenceof the agent, wherein an increase in the expression of the reporterprotein in the presence of the agent as compared to the expression ofthe protein in the absence of the agent identifies the agent as acompound that increases the expression of the protein.

Also disclosed are methods of identifying a compound that increasesexpression of a protein by: (i) providing a cell containing a reporterconstruct containing (a) a promoter sequence of a gene encoding DIP5,LST8, REG1, UGP1, SEC21, SEC28, SFT1, IME2, KSP1, RCK2, YCK3, LSG1,MGA2, MUM2, OSH3, PIN4, URE2, PTC4, NTH1, CUP9, JSN1, TIF4632, HRD1,SAN1, YBR030w, YKL088w, YML081w, YMR111c, YOR291W, YOR062C, GIS3, PTP2,SKO1, HAP4, VHR1, SUM1, STB3, PFS1, OSH2, ISN1, SLC7A2, SLC7A13, SLC7A6,GBL, DSSP, UGP2, COPG, COPG2, COPE, BET1, ICK, MAK, ULK3, MARK2, MARK1,MARK3, MARK4, ULK1, CAMK1G, CAMK1, CAMK1D, CSNK1G3, CSNK1G2, CSNK1G1,CSNK1D1, CSNK1E1, CSNK1A1, CSNK1A1L, LSG1, OSBPL1A, ERC2, OSBPL6,OSBPL3, OSBPL7, OSBPL1A, OSBPL2, CFTF2T, CFTF2, EEF1G, PPM1G, PPM1A,PPM1B, TREH, PKNOX2, PKNOX1, PUM1, PUM2, EIF4G3, EIF4G1, SYVN1, ZNF364,SETD6, PPCDC, EGR3, ZFP161, EGR2, HKR1, ZNF740, ATP13A2 (PARK9),ATP13A1, ATP13A3, ATP13A4, or ATP13A5, and (b) a nucleotide sequenceencoding a reporter protein; (ii) contacting the cell with an agent; and(iii) measuring the expression of the reporter protein in the presenceof the agent, wherein an increase in the expression of the reporterprotein in the presence of the agent as compared to the expression ofthe protein in the absence of the agent identifies the agent as acompound that increases the expression of the protein.

Also disclosed are methods of identifying a compound that increases theactivity of a protein by: (i) providing a protein selected from thegroup consisting of QDR3, DIP5, LST8, REG1, UGP1, SEC21, SEC28, SFT1,IME2, KSP1, RCK2, YCK3, LSG1, MGA2, MUM2, OSH3, PIN4, URE2, PTC4, NTH1,CUP9, JSN1, TIF4632, HRD1, SAN1, YBR030w, YKL088w, YML081w, YMR111c,YOR291W, YOR062C, GIS3, PTP2, SKO1, HAP4, VHR1, SUM1, STB3, PFS1, OSH2,ISN1, SLC7A2, SLC7A13, SLC7A6, GBL, DSSP, UGP2, COPG, COPG2, COPE, BET1,ICK, MAK, ULK3, MARK2, MARK1, MARK3, MARK4, ULK1, CAMK1G, CAMK1, CAMK1D,CSNK1G3, CSNK1G2, CSNK1G1, CSNK1D1, CSNK1E1, CSNK1A1, CSNK1A1L, LSG1,OSBPL1A, ERC2, OSBPL6, OSBPL3, OSBPL7, OSBPL1A, OSBPL2, CFTF2T, CFTF2,EEF1G, PPM1G, PPM1A, PPM1B, TREH, PKNOX2, PKNOX1, PUM1, PUM2, EIF4G3,EIF4G1, SYVN1, ZNF364, SETD6, PPCDC, EGR3, ZFP161, EGR2, HKR1, ZNF740,ATP (PARK9), ATP13A1, ATP13A3, ATP13A4, and ATP13A5; (ii) contacting theprotein with an agent; and (iii) measuring the activity of the proteinin the presence of the agent, wherein an increase in the activity of theprotein in the presence of the agent as compared to the activity of theprotein in the absence of the agent identifies the agent as a compoundthat increases the activity the protein.

Also disclosed are methods of identifying a compound that increases theactivity of a protein by: (i) providing a protein selected from thegroup consisting of DIP5, LST8, REG1, UGP1, SEC21, SEC28, SFT1, IME2,KSP1, RCK2, YCK3, LSG1, MGA2, MUM2, OSH3, PIN4, URE2, PTC4, NTH1, CUP9,JSN1, TIF4632, HRD1, SAN1, YBR030w, YKL088w, YML081w, YMR111c, YOR291W,YOR062C, GIS3, PTP2, SKO1, HAP4, VHR1, SUM1, STB3, PFS1, OSH2, ISN1,SLC7A2, SLC7A13, SLC7A6, GBL, DSSP, UGP2, COPG, COPG2, COPE, BET1, ICK,MAK, ULK3, MARK2, MARK1, MARK3, MARK4, ULK1, CAMK1G, CAMK1, CAMK1D,CSNK1G3, CSNK1G2, CSNK1G1, CSNK1D1, CSNK1E1, CSNK1A1, CSNK1A1L, LSG1,OSBPL1A, ERC2, OSBPL6, OSBPL3, OSBPL7, OSBPL1A, OSBPL2, CFTF2T, CFTF2,EEF1G, PPM1G, PPM1A, PPM1B, TREH, PKNOX2, PKNOX1, PUM1, PUM2, EIF4G3,EIF4G1, SYVN1, ZNF364, SETD6, PPCDC, EGR3, ZFP161, EGR2, HKR1, ZNF740,ATP13A2 (PARK9), ATP13A1, ATP13A3, ATP13A4, and ATP13A5; (ii) contactingthe protein with an agent; and (iii) measuring the activity of theprotein in the presence of the agent, wherein an increase in theactivity of the protein in the presence of the agent as compared to theactivity of the protein in the absence of the agent identifies the agentas a compound that increases the activity the protein.

In the foregoing methods, the agent can be a synthetic compound or anaturally occurring compound. For example, the agent can be a smallmolecule, nucleic acid, protein, antibody, or peptidomimetic. The cellused in the methods can be a eukaryotic cell (e.g., a yeast cell,nematode, insect, or mammalian cell), a prokaryotic cell, or a cellobtained from an alpha-synuclein transgenic animal.

Also disclosed are methods of evaluating an individual for the presenceof or susceptibility to developing a synucleinopathy. The methodsinclude the steps of: obtaining a biological sample from a firstsubject; analyzing the sample for the expression or activity of one ormore proteins selected from the group consisting of SLC7A2, SLC7A13,SLC7A6, GBL, DSSP, UGP2, COPG, COPG2, COPE, BET1, ICK, MAK, ULK3, MARK2,MARK1, MARK3, MARK4, ULK1, CAMK1G, CAMK1, CAMK1D, CSNK1G3, CSNK1G2,CSNK1G1, CSNK1D1, CSNK1E1, CSNK1A1, CSNK1A1L, LSG1, OSBPL1A, ERC2,OSBPL6, OSBPL3, OSBPL7, OSBPL1A, OSBPL2, CFTF2T, CFTF2, EEF1G, PPM1G,PPM1A, PPM1B, TREH, PKNOX2, PKNOX1, PUM1, PUM2, EIF4G3, EIF4G1, SYVN1,ZNF364, SETD6, PPCDC, EGR3, ZFP161, EGR2, HKR1, ZNF740, ATP13A2 (PARKS),ATP13A1, ATP13A3, ATP13A4, and ATP13A5; and comparing the expression oractivity of the one or more proteins in the sample from the firstsubject with the expression or activity of the one or more proteins in asample from a second subject not having or being at risk of developingthe synucleinopathy, wherein decreased expression or activity of the oneor more proteins in the sample from the first subject indicates that thesubject is an individual having or at risk of developing thesynucleinopathy.

Also disclosed are methods of evaluating an individual for the presenceof or susceptibility to developing a synucleinopathy. The methodsinclude the steps of: obtaining a biological sample from a firstsubject; analyzing the sample for the expression or activity of one ormore proteins selected from the group consisting of GOSR1, SEC31A,SEC31B, ADIPOR2, ADIPOR1, KLF12, KLF5, ZNF323, ZNF718, ZNF705A, RABGGTA,ZNF289, SLC35E1, NIBP, RABAC1, KIAA0999, PPP1CC, PPP1CB, PPP1CA, USP21,and USP2; and comparing the expression or activity of the one or moreproteins in the sample from the first subject with the expression oractivity of the one or more proteins in a sample from a second subjectnot having or being at risk of developing the synucleinopathy, whereinincreased expression or activity of the one or more proteins in thesample from the first subject indicates that the subject is anindividual having or at risk of developing the synucleinopathy.

Also disclosed are methods of evaluating an individual for the presenceof or susceptibility to developing a synucleinopathy. The methodsinclude the steps of: obtaining a biological sample from a firstsubject; analyzing the sample for the expression or activity of one ormore proteins selected from the group consisting of KLF12, KLF5, ZNF323,ZNF718, ZNF705A, RABGGTA, ZNF289, SLC35E1, NIBP, RABAC1, KIAA0999,PPP1CC, PPP1CB, PPP1CA, USP21, and USP2; and comparing the expression oractivity of the one or more proteins in the sample from the firstsubject with the expression or activity of the one or more proteins in asample from a second subject not having or being at risk of developingthe synucleinopathy, wherein increased expression or activity of the oneor more proteins in the sample from the first subject indicates that thesubject is an individual having or at risk of developing thesynucleinopathy.

Also disclosed are pharmaceutical compositions containing a therapeuticor prophylactic amount of a compound that: (i) increases the expressionor activity of SLC7A2, SLC7A13, SLC7A6, GBL, DSSP, UGP2, COPG, COPG2,COPE, BET1, ICK, MAK, ULK3, MARK2, MARK1, MARK3, MARK4, ULK1, CAMK1G,CAMK1, CAMK1D, CSNK1G3, CSNK1G2, CSNK1G1, CSNK1D1, CSNK1E1, CSNK1A1,CSNK1A1L, LSG1, OSBPL1A, ERC2, OSBPL6, OSBPL3, OSBPL7, OSBPL1A, OSBPL2,CFTF2T, CFTF2, EEF1G, PPM1G, PPM1A, PPM1B, TREH, PKNOX2, PKNOX1, PUM1,PUM2, EIF4G3, EIF4G1, SYVN1, ZNF364, SETD6, PPCDC, EGR3, ZFP161, EGR2,HKR1, ZNF740, ATP13A2 (PARK9), ATP13A1, ATP13A3, ATP13A4, or ATP13A5; or(ii) decreases the expression or activity of GOSR1, SEC31A, SEC31B,ADIPOR2, ADIPOR1, KLF12, KLF5, ZNF323, ZNF718, ZNF705A, RABGGTA, ZNF289,SLC35E1, NIBP, RABAC1, KIAA0999, PPP1CC, PPP1CB, PPP1CA, USP21, or USP2.

Also disclosed are pharmaceutical compositions containing a therapeuticor prophylactic amount of a compound that: (i) increases the expressionor activity of SLC7A2, SLC7A13, SLC7A6, GBL, DSSP, UGP2, COPG, COPG2,COPE, BET1, ICK, MAK, ULK3, MARK2, MARK1, MARK3, MARK4, ULK1, CAMK1G,CAMK1, CAMK1D, CSNK1G3, CSNK1G2, CSNK1G1, CSNK1D1, CSNK1E1, CSNK1A1,CSNK1A1L, LSG1, OSBPL1A, ERC2, OSBPL6, OSBPL3, OSBPL7, OSBPL1A, OSBPL2,CFTF2T, CFTF2, EEF1G, PPM1G, PPM1A, PPM1B, TREH, PKNOX2, PKNOX1, PUM1,PUM2, EIF4G3, EIF4G1, SYVN1, ZNF364, SETD6, PPCDC, EGR3, ZFP161, EGR2,HKR1, ZNF740, ATP13A2 (PARKS), ATP13A1, ATP13A3, ATP13A4, or ATP13A5; or(ii) decreases the expression or activity of KLF12, KLF5, ZNF323,ZNF718, ZNF705A, RABGGTA, ZNF289, SLC35E1, NIBP, RABAC1, KIAA0999,PPP1CC, PPP1CB, PPP1CA, USP21, or USP2.

In some embodiments, the pharmaceutical composition contains (i) atherapeutic or prophylactic amount of an isolated polypeptide selectedfrom the group consisting of SLC7A2, SLC7A13, SLC7A6, GBL, DSSP, UGP2,COPG, COPG2, COPE, BET1, ICK, MAK, ULK3, MARK2, MARK1, MARK3, MARK4,ULK1, CAMK1G, CAMK1, CAMK1D, CSNK1G3, CSNK1G2, CSNK1G1, CSNK1D1,CSNK1E1, CSNK1A1, CSNK1A1L, LSG1, OSBPL1A, ERC2, OSBPL6, OSBPL3, OSBPL7,OSBPL1A, OSBPL2, CFTF2T, CFTF2, EEF1G, PPM1G, PPM1A, PPM1B, TREH,PKNOX2, PKNOX1, PUM1, PUM2, EIF4G3, EIF4G1, SYVN1, ZNF364, SETD6, PPCDC,EGR3, ZFP161, EGR2, HKR1, ZNF740, ATP13A2 (PARK9), ATP13A1, ATP13A3,ATP13A4, and ATP13A5, and (ii) a pharmaceutically acceptable carrier.

Also disclosed are cells containing a first expression vector encodingalpha synuclein and a second expression vector encoding GOS1, SEC31,IZH3, MKS1, TPO4, GOSR1, SEC31A, SEC31B, ADIPOR2, ADIPOR1, NRG1, NRG2,BET4, GLO3, SLY41, TRS120, YIP3, IDS2, PPZ2, UBP11, UBP7, SIP5,MATALPHA1, KLF12, KLF5, ZNF323, ZNF718, ZNF705A, RABGGTA, ZNF289,SLC35E1, NIBP, RABAC1, KIAA0999, PPP1CC, PPP1CB, PPP1CA, USP21, or USP2.The cell can be a eukaryotic cell (e.g., a yeast cell or mammalian cell)or a prokaryotic cell.

Also disclosed are cells containing a first expression vector encodingalpha synuclein and a second expression vector encoding NRG1, NRG2,BET4, GLO3, SLY41, TRS120, YIP3, IDS2, PPZ2, UBP11, UBP7, SIP5,MATALPHA1, KLF12, KLF5, ZNF323, ZNF718, ZNF705A, RABGGTA, ZNF289,SLC35E1, NIBP, RABAC1, KIAA0999, PPP1CC, PPP1CB, PPP1CA, USP21, or USP2.The cell can be a eukaryotic cell (e.g., a yeast cell or mammalian cell)or a prokaryotic cell.

Also disclosed are cells containing a first expression vector encodingalpha synuclein and a second expression vector encoding QDR3, DIP5,LST8, REG1, UGP1, SEC21, SEC28, SFT1, IME2, KSP1, RCK2, YCK3, LSG1,MGA2, MUM2, OSH3, PIN4, URE2, PTC4, NTH1, CUP9, JSN1, TIF4632, HRD1,SAN1, YBR030w, YKL088w, YML081w, YMR111c, YOR291W, YOR062C, GIS3, PTP2,SKO1, HAP4, VHR1, SUM1, STB3, PFS1, OSH2, ISN1, SLC7A2, SLC7A13, SLC7A6,GBL, DSSP, UGP2, COPG, COPG2, COPE, BET1, ICK, MAK, ULK3, MARK2, MARK1,MARK3, MARK4, ULK1, CAMK1G, CAMK1, CAMK1D, CSNK1G3, CSNK1G2, CSNK1G1,CSNK1D1, CSNK1E1, CSNK1A1, CSNK1A1L, LSG1, OSBPL1A, ERC2, OSBPL6,OSBPL3, OSBPL7, OSBPL1A, OSBPL2, CFTF2T, CFTF2, EEF1G, PPM1G, PPM1A,PPM1B, TREH, PKNOX2, PKNOX1, PUM1, PUM2, EIF4G3, EIF4G1, SYVN1, ZNF364,SETD6, PPCDC, EGR3, ZFP161, EGR2, HKR1, ZNF740, ATP13A2 (PARK9),ATP13A1, ATP13A3, ATP13A4, or ATP13A5. The cell can be a eukaryotic cell(e.g., a yeast cell or mammalian cell) or a prokaryotic cell.

Also disclosed are cells containing a first expression vector encodingalpha synuclein and a second expression vector encoding DIP5, LST8,REG1, UGP1, SEC21, SEC28, SFT1, IME2, KSP1, RCK2, YCK3, LSG1, MGA2,MUM2, OSH3, PIN4, URE2, PTC4, NTH1, CUP9, JSN1, TIF4632, HRD1, SAN1,YBR030w, YKL088w, YML081w, YMR111c, YOR291W, YOR062C, GIS3, PTP2, SKO1,HAP4, VHR1, SUM1, STB3, PFS1, OSH2, ISN1, SLC7A2, SLC7A13, SLC7A6, GBL,DSSP, UGP2, COPG, COPG2, COPE, BET1, ICK, MAK, ULK3, MARK2, MARK1,MARK3, MARK4, ULK1, CAMK1G, CAMK1, CAMK1D, CSNK1G3, CSNK1G2, CSNK1G1,CSNK1D1, CSNK1E1, CSNK1A1, CSNK1A1L, LSG1, OSBPL1A, ERC2, OSBPL6,OSBPL3, OSBPL7, OSBPL1A, OSBPL2, CFTF2T, CFTF2, EEF1G, PPM1G, PPM1A,PPM1B, TREH, PKNOX2, PKNOX1, PUM1, PUM2, EIF4G3, EIF4G1, SYVN1, ZNF364,SETD6, PPCDC, EGR3, ZFP161, EGR2, HKR1, ZNF740, ATP13A2 (PARK9),ATP13A1, ATP13A3, ATP13A4, or ATP13A5. The cell can be a eukaryotic cell(e.g., a yeast cell or mammalian cell) or a prokaryotic cell.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentapplication, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DETAILED DESCRIPTION

It has been found that overexpression of certain genes results in amodulation of alpha-synuclein mediated cellular toxicity. Compounds thatmodulate expression of these genes or activity of the encoded proteinscan be used to inhibit alpha-synuclein mediated toxicity and used totreat or prevent synucleinopathies such as Parkinson's disease.

Modulators of Alpha-Synuclein-Mediated Toxicity

As detailed in the accompanying examples, several genes have beenidentified that modulate cellular toxicity associated withoverexpression of alpha-synuclein in yeast cells. For those genes thatwere found to suppress toxicity when overexpressed in yeast, it isexpected that enhancing expression of the genes and/or activity ofproteins encoded by the genes will result in a suppression of toxicityin alpha-synuclein expressing cells. Conversely, for those genes thatwere found to enhance toxicity when overexpressed in yeast, it isexpected that inhibiting expression of the genes and/or the activity ofproteins encoded by the genes will result in a suppression of toxicityin alpha-synuclein expressing cells.

It is expected that the mechanisms by which alpha-synuclein inducestoxicity in the yeast model system described herein is similar to themechanisms by which alpha-synuclein induces toxicity in human cells.Many of the yeast genes identified as modulating alpha-synucleinmediated toxicity in yeast cells have orthologous or highly relatedgenes in humans (Table 1). As a result, human counterparts of theidentified yeast genes are expected to be useful targets for modulatingalpha-synuclein mediated toxicity in human cells.

Table 1 lists GenBank™ Accession Numbers corresponding to the nucleotideand protein sequences for each of the human genes identified herein. Asdetailed in the following sections, these nucleotide and proteinsequences can be used to generate compounds (including but not limitedto nucleic acids, peptides, antibodies) that modulate expression ofgenes or activity of encoded gene products. The genes identified hereinas modulators of alpha-synuclein mediated toxicity are referred to insubsequent sections (e.g., regarding screening assays) as “target genes”and the encoded proteins are referred to as “target proteins.”

TABLE 1 Human Counterparts of Yeast Genes that Modulate Alpha-SynucleinToxicity Yeast Gene Suppressor or Human Gene DNA Accession ProteinAccession Name Enhancer Name Number (Human) Number (Human) DIP5Suppressor SLC7A2 NM_003046 NP_003037 SLC7A13 NM_138817 NP_620172 SLC7A6NM_001076785 NP_001070253 LST8 Suppressor GBL NM_022372 NP_071767 REG1Suppressor DSSP NM_014208 NP_055023 UGP1 Suppressor UGP2 NM_006759NP_006750 SEC21 Suppressor COPG NM_016128 NP_057212 COPG2 NM_012133NP_036265 SEC28 Suppressor COPE NM_007263 NP_009194 SFT1 Suppressor BET1NM_005868 NP_005859 IME2 Suppressor ICK NM_016513 NP_057597 MAKNM_005906 NP_005897 KSP1 Suppressor ULK3 AL117482 CAB55955 MARK2NM_001039469 NP_001034558 MARK1 NM_018650 NP_061120 MARK3 NM_002376NP_002367 MARK4 NM_031417 NP_113605 ULK1 NM_003565 NP_003556 RCK2Suppressor CAMK1G NM_020439 NP_065172 CAMK1 NM_003656 NP_003647 CAMK1DNM_153498 NP_705718 YCK3 Suppressor CSNK1G3 NM_001044723 NP_001038188CSNK1G2 NM_001319 NP_001310 CSNK1G1 NM_022048 NP_071331 CSNK1D1NM_001893 NP_001884 CSNK1E1 NM_152221 NP_689407 CSNK1A1 NM_001025105NP_001020276 CSNK1A1L NM_145203 NP_660204 LSG1 Suppressor LSG1 NM_018385NP_060855 MGA2 Suppressor OSBPL1A NM_080597 NP_542164 MUM2 SuppressorERC2 BC111550 AAI11551 OSH3 Suppressor OSBPL6 NM_145739 NP_665682 OSBPL3NM_015550 NP_056365 OSBPL7 NM_145798 NP_665741 OSBPL1A NM_080597NP_542164 OSBPL2 NM_144498 NP_653081 PIN4 Suppressor CFTF2T NM_015235NP_056050 CFTF2 NM_001325 NP_001316 URE2 Suppressor EEF1G CR407625CAG28553 PTC4 Suppressor PPM1G NM_177983 NP_817092 PPM1A NM_177952NP_808821 PPM1B NM_002706 NP_002697 NTH1 Suppressor TREH NM_007180NP_009111 CUP9 Suppressor PKNOX2 NM_022062 NP_071345 PKNOX1 NM_004571NP_004562 JSN1 Suppressor PUM1 NM_001020658 NP_001018494 PUM2 NM_015317NP_056132 TIF4632 Suppressor EIF4G3 NM_003760 NP_003751 EIF4G1 NM_198241NP_937884 HRD1 Suppressor SYVN1 NM_172230 NP_757385 SAN1 SuppressorZNF364 NM_014455 NP_055270 YBR030w Suppressor SETD6 BC022451 AAH22451YKL088w Suppressor PPCDC NM_021823 NP_068595 YML081w Suppressor EGR3NM_004430 NP_004421 ZFP161 NM_003409 NP_003400 EGR2 NM_000399 NP_000390HKR1 NM_181786 NP_861451 ZNF740 NM_001004304 NP_001004304 YOR291WSuppressor ATP13A2 NM_022089 NP_071372 (PARK9) ATP13A1 NM_020410NP_065143 ATP13A3 NM_024524 NP_078800 ATP13A4 NM_032279 NP_115655ATP13A5 NM_198505 NP_940907 GOS1 Enhancer GOSR1 BC012620 AAH12620 SEC31Enhancer SEC31A BC047883 AAH47883 SEC31B NM_015490 NP_056305 IZH3Enhancer ADIPOR2 NM_024551 NP_078827 ADIPOR1 NM_001127687 NP_001121159NRG1 Enhancer KLF12 NM_007249 NP_009180 KLF5 NM_001730 NP_001721 NRG2Enhancer ZNF323 NM_145909 NP_665916 ZNF718 NM_001039127 NP_001034216ZNF705A NM_001004328 NP_001004328 BET4 Enhancer RABGGTA NM_004581NP_004572 GLO3 Enhancer ZNF289 NM_032389 NP_115765 SLY41 EnhancerSLC35E1 NM_024881 NP_079157 TRS120 Enhancer NIBP BC065288 AAH65288 YIP3Enhancer RABAC1 NM_006423 NP_006414 IDS2 Enhancer KIAA0999 AB023216BAA76843 PPZ2 Enhancer PPP1CC NM_002710 NP_002701 PPP1CB NM_206876NP_996759 PPP1CA NM_001008709 NP_001008709 UBP11 Enhancer USP21NM_012475 NP_036607 UBP7 Enhancer USP21 NM_012475 NP_036607 USP2NM_004205 NP_004196

Compounds that enhance the expression or activity of DIPS (or humanSLC7A2, SLC7A13, or SLC7A6), LST8 (or human GBL), REG1 (or human DSSP),UGP1 (or human UGP2), SEC21 (or human COPG or COPG2), SEC28 (or humanCOPE), SFT1 (or human BET1), IME2 (or human ICK or MAK), KSP1 (or humanULK3, MARK2, MARK1, MARK3, MARK4, or ULK1), RCK2 (or human CAMK1G,CAMK1, or CAMK1D), YCK3 (or human CSNK1G3, CSNK1G2, CSNK1G1, CSNK1D1,CSNK1E1, CSNK1A1, or CSNK1A1L), LSG1 (or human LSG1), MGA2 (or humanOSBPL1A), MUM2 (or human ERC2), OSH3 (or human OSBPL6, OSBPL3, OSBPL7,OSBPL1A, or OSBPL2), PIN4 (or human CFTF2T or CFTF2), URE2 (or humanEEF1G), PTC4 (or human PPM1G PPM1A, or PPM1B), NTH1 (or human TREH),CUP9 (or human PKNOX2 or PKNOX1), JSN1 (or human PUM1 or PUM2), TIF4632(or human EIF4G3 or EIF4G1), HRD1 (or human SYVN1), SAN1 (or humanZNF364), YBR030w (or human SETD6), YKL088w (or human PPCDC), or YOR291W(or human ATP13A2 (PARK9), ATP13A1, ATP13A3, ATP13A4, or ATP 13A5) areexpected to inhibit alpha-synuclein-mediated cellular toxicity. It isalso understood that compounds capable of inhibiting the expression oractivity of an inhibitor of DIP5, LST8, REG1, UGP1, SEC21, SEC28, SFT1,IME2, KSP1, RCK2, YCK3, LSG1, MGA2, MUM2, OSH3, PIN4, URE2, PTC4, NTH1,CUP9, JSN1, TIF4632, HRD1, SAN1, YBR030w, YKL088w, YMR111c, YOR291W, orhuman counterparts of any of the foregoing are expected to inhibitalpha-synuclein-mediated cellular toxicity.

Alternatively, compounds that inhibit the expression of GOS1 (or humanGOSR1), SEC31 (or human SEC31A or SEC31B), IZH3 (or human ADIPOR2 orADIPOR1), NRG1 (or human KLF12 or KLF5), NRG2 (or human ZNF323, ZNF718,or ZNF705A), BET4 (or human RABGGTA), GLO3 (or human ZNF289), SLY41 (orhuman SLC35E1), TRS120 (or human NIBP), YIP3 (or human RABAC1), IDS2 (orhuman KIAA0999), PPZ2 (or human PPP1CC, PPP1CB, or PPP1CA), UBP11 (orhuman USP21), or UBP7 (or human USP21 or USP2) are expected to inhibitalpha-synuclein-mediated cellular toxicity. It is also understood thatcompounds capable of enhancing the expression or activity of aninhibitor of GOS1, SEC31, IZH3, NRG1, NRG2, BET4, GLO3, SLY41, TRS120,YIP3, IDS2, PPZ2, UBP11, UBP7, or human counterparts of any of theforegoing are expected to inhibit alpha-synuclein-mediated cellulartoxicity.

Screening Assays

The methods described herein include methods (also referred to herein as“screening assays”) for identifying compounds that modulate (i.e.,increase or decrease) expression or activity of selected target genes ortheir protein products. Such compounds include, e.g., polypeptides,peptides, antibodies, peptidomimetics, peptoids, small inorganicmolecules, small non-nucleic acid organic molecules, nucleic acids(e.g., anti-sense nucleic acids, siRNA, oligonucleotides, syntheticoligonucleotides), carbohydrates, or other agents that bind to thetarget proteins, have a stimulatory or inhibitory effect on, forexample, expression of a target gene or activity of a target protein.Compounds thus identified can be used to modulate the expression oractivity of target genes or target proteins in a therapeutic protocol.

In general, screening assays involve assaying the effect of a test agenton expression or activity of a target nucleic acid or target protein ina test sample (i.e., a sample containing the target nucleic acid ortarget protein). Expression or activity in the presence of the testcompound or agent can be compared to expression or activity in a controlsample (i.e., a sample containing the target protein that is incubatedunder the same conditions, but without the test compound). A change inthe expression or activity of the target nucleic acid or target proteinin the test sample compared to the control indicates that the test agentor compound modulates expression or activity of the target nucleic acidor target protein and is a candidate agent.

Compounds can be tested for their ability to modulate one or moreactivities mediated by a target protein described herein. For example,compounds that modulate expression of a gene or activity of a proteinlisted in Table 1 or 2 can be tested for their ability to modulatetoxicity in cells expressing alpha-synuclein. Methods of assaying acompound for such activities are known in the art. In some cases, acompound is tested for it's ability to directly affect target geneexpression or binding to a target protein (e.g., by decreasing theamount of target RNA in a cell or decreasing the amount of targetprotein in a cell) and tested for its ability to modulate a metaboliceffect associated with the target protein.

In one embodiment, assays are provided for screening candidate or testmolecules that are substrates of a target protein or a biologicallyactive portion thereof in a cell. In another embodiment, the assays arefor screening candidate or test compounds that bind to a target proteinor modulate the activity of a target protein or a biologically activeportion thereof. Such compounds include those that disrupt theinteraction between a target protein and its ligand.

The test compounds used in the methods can be obtained using any of thenumerous approaches in the art including combinatorial library methods,including: biological libraries; peptoid libraries (libraries ofmolecules having the functionalities of peptides, but with a novel,non-peptide backbone which are resistant to enzymatic degradation butwhich nevertheless remain bioactive; e.g., Zuckermann et al. (1994) J.Med. Chem. 37:2678); spatially addressable parallel solid phase orsolution phase libraries; synthetic library methods requiringdeconvolution; the “one-bead one-compound” library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary and peptoid library approaches are limited to peptide libraries,while the other four approaches are applicable to peptide, non-peptideoligomer or small molecule libraries of compounds (Lam (1997) AnticancerDrug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the literature, for example in: DeWitt et al., Proc. Natl.Acad. Sci. USA, 90:6909, 1993; Erb et al., Proc. Natl. Acad. Sci. USA,91:11422, 1994; Zuckermann et al., J. Med. Chem. 37:2678, 1994; Cho etal., Science 261:1303, 1993; Carrell et al., Angew. Chem. Int. Ed. Engl.33:2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl., 33:2061,1994; and Gallop et al., J. Med. Chem., 37:1233, 1994.

Libraries of compounds may be presented in solution (e.g., Houghten,Bio/Techniques, 13:412421,1992), or on beads (Lam, Nature, 354:82-84,1991), chips (Fodor, Nature 364:555-556, 1993), bacteria (U.S. Pat. No.5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and 5,223,409),plasmids (Cull et al., Proc. Natl. Acad. Sci. USA, 89:1865-1869, 1992)or phage (Scott and Smith, Science, 249:386-390, 1990; Devlin, Science,249:404-406, 1990; Cwirla et al., Proc. Natl. Acad. Sci. USA,87:6378-6382, 1990; and Felici, J. Mol. Biol., 222:301-310, 1991).

In one embodiment, a cell-based assay is employed in which a cell thatexpresses a target protein or biologically active portion thereof iscontacted with a test compound. The ability of the test compound tomodulate expression or activity of the target protein is thendetermined. The cell, for example, can be a yeast cell or a cell ofmammalian origin, e.g., rat, mouse, or human.

The ability of the test compound to bind to a target protein or modulatetarget protein binding to a compound, e.g., a target protein substrate,can also be evaluated. This can be accomplished, for example, bycoupling the compound, e.g., the substrate, with a radioisotope orenzymatic label such that binding of the compound, e.g., the substrate,to the target protein can be determined by detecting the labeledcompound, e.g., substrate, in a complex. Alternatively, the targetprotein can be coupled with a radioisotope or enzymatic label to monitorthe ability of a test compound to modulate target protein binding to atarget protein substrate in a complex. For example, compounds (e.g.,target protein substrates) can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H,either directly or indirectly, and the radioisotope detected by directcounting of radioemmission or by scintillation counting. Alternatively,compounds can be enzymatically labeled with, for example, horseradishperoxidase, alkaline phosphatase, or luciferase, and the enzymatic labeldetected by determination of conversion of an appropriate substrate toproduct.

The ability of a compound (e.g., a target protein substrate) to interactwith target protein with or without the labeling of any of theinteractants can be evaluated. For example, a microphysiometer can beused to detect the interaction of a compound with a target proteinwithout the labeling of either the compound or the target protein(McConnell et al., Science 257:1906-1912, 1992). As used herein, a“microphysiometer” (e.g., Cytosensor™) is an analytical instrument thatmeasures the rate at which a cell acidifies its environment using alight-addressable potentiometric sensor (LAPS). Changes in thisacidification rate can be used as an indicator of the interactionbetween a compound and a target protein.

In yet another embodiment, a cell-free assay is provided in which atarget protein or biologically active portion thereof is contacted witha test compound and the ability of the test compound to bind to thetarget protein or biologically active portion thereof is evaluated. Ingeneral, biologically active portions of target proteins to be used inassays described herein include fragments that participate ininteractions with other molecules, e.g., fragments with high surfaceprobability scores.

Cell-free assays involve preparing a reaction mixture of the targetprotein and the test compound under conditions and for a time sufficientto allow the two components to interact and bind, thus forming a complexthat can be removed and/or detected.

The interaction between two molecules can also be detected usingfluorescence energy transfer (FET) (see, for example, Lakowicz et al.,U.S. Pat. No. 5,631,169; Stavrianopoulos et al., U.S. Pat. No.4,868,103). A fluorophore label on the first, “donor” molecule isselected such that its emitted fluorescent energy will be absorbed by afluorescent label on a second, “acceptor” molecule, which in turn isable to fluoresce due to the absorbed energy. Alternately, the “donor”protein molecule may use the natural fluorescent energy of tryptophanresidues. Labels are chosen that emit different wavelengths of light,such that the “acceptor” molecule label may be differentiated from thatof the “donor.” Since the efficiency of energy transfer between thelabels is related to the distance separating the molecules, the spatialrelationship between the molecules can be assessed. In a situation inwhich binding occurs between the molecules, the fluorescent emission ofthe “acceptor” molecule label in the assay should be maximal. A FETbinding event can be conveniently measured through standard fluorometricdetection means well known in the art (e.g., using a fluorimeter).

In another embodiment, the ability of a target protein to bind to atarget molecule can be determined using real-time BiomolecularInteraction Analysis (BIA) (e.g., Sjolander et al., Anal. Chem.,63:2338-2345, 1991, and Szabo et al., Curr. Opin. Struct. Biol.,5:699-705, 1995). “Surface plasmon resonance” or “BIA” detectsbiospecific interactions in real time, without labeling any of theinteractants (e.g., BlAcore). Changes in the mass at the binding surface(indicative of a binding event) result in alterations of the refractiveindex of light near the surface (the optical phenomenon of surfaceplasmon resonance (SPR)), resulting in a detectable signal which can beused as an indication of real-time reactions between biologicalmolecules.

In various of these assays, the target protein or the test substance isanchored onto a solid phase. The target protein/test compound complexesanchored on the solid phase can be detected at the end of the reaction.Generally, the target protein is anchored onto a solid surface, and thetest compound (which is not anchored) can be labeled, either directly orindirectly, with detectable labels discussed herein.

It may be desirable to immobilize either the target protein, ananti-target protein antibody, or its target molecule to facilitateseparation of complexed from uncomplexed forms of one or both of theproteins, as well as to accommodate automation of the assay. Binding ofa test compound to a target protein, or interaction of a target proteinwith a target molecule in the presence and absence of a test compound,can be accomplished in any vessel suitable for containing the reactants.Examples of such vessels include microtiter plates, test tubes, andmicro-centrifuge tubes. In one embodiment, a fusion protein can beprovided that adds a domain that allows one or both of the proteins tobe bound to a matrix. For example, glutathione-S-transferase/targetprotein fusion proteins or glutathione-S-transferase/target fusionproteins can be adsorbed onto glutathione Sepharose™ beads (SigmaChemical, St. Louis, Mo.) or glutathione derivatized microtiter plates,which are then combined with the test compound or the test compound andeither the non-adsorbed target protein. The mixture is then incubatedunder conditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotiter plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, and the complex determinedeither directly or indirectly, for example, as described above.Alternatively, the complexes can be dissociated from the matrix, and thelevel of target protein binding or activity determined using standardtechniques.

Other techniques for immobilizing a target protein on matrices includeusing conjugation of biotin and streptavidin. Biotinylated targetprotein can be prepared from biotin-NHS (N-hydroxy-succinimide) usingtechniques known in the art (e.g., biotinylation kit, Pierce Chemicals,Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96well plates (Pierce Chemical).

To conduct the assay, the non-immobilized component is added to thecoated surface containing the anchored component. After the reaction iscomplete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The complexes anchored on the solid surface can bedetected in a number of ways. Where the previously non-immobilizedcomponent is pre-labeled, the presence of a label immobilized on thesurface indicates that complexes were formed. Where the previouslynon-immobilized component is not pre-labeled, an indirect label can beused to detect complexes anchored on the surface; e.g., using a labeledantibody specific for the immobilized component (the antibody, in turn,can be directly labeled or indirectly labeled with, e.g., a labeledanti-Ig antibody).

In some cases, the assay is performed utilizing antibodies reactive withtarget protein, but which do not interfere with binding of the targetprotein to its target molecule. Such antibodies can be derivatized tothe wells of the plate, and unbound target protein trapped in the wellsby antibody conjugation. Methods for detecting such complexes, inaddition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with thetarget protein or target molecule, as well as enzyme-linked assays whichrely on detecting an enzymatic activity associated with the targetprotein.

Alternatively, cell-free assays can be conducted in a liquid phase. Insuch an assay, the reaction products are separated from unreactedcomponents, by any of a number of standard techniques, including but notlimited to: differential centrifugation (see, for example, Rivas andMinton, Trends Biochem. Sci., 18:284-7, 1993); chromatography (gelfiltration chromatography, ion-exchange chromatography); electrophoresis(e.g., Ausubel et al., eds. Current Protocols in Molecular Biology 1999,J. Wiley: New York.); and immunoprecipitation (see, for example, Ausubelet al., eds., 1999, Current Protocols in Molecular Biology, J. Wiley:New York). Such resins and chromatographic techniques are known to oneskilled in the art (e.g., Heegaard, J. Mol. Recognit., 11: 141-148,1998; Hage et al., J. Chromatogr. B. Biomed. Sci. Appl., 699:499-525,1997). Further, fluorescence energy transfer may also be convenientlyutilized, as described herein, to detect binding without furtherpurification of the complex from solution.

The assay can include contacting the target protein or a biologicallyactive portion thereof with a known compound that binds to the targetprotein to form an assay mixture, contacting the assay mixture with atest compound, and determining the ability of the test compound tointeract with the target protein, wherein determining the ability of thetest compound to interact with the target protein includes determiningthe ability of the test compound to preferentially bind to the targetprotein or biologically active portion thereof, or to modulate theactivity of a target molecule, as compared to the known compound.

A target protein can, in vivo, interact with one or more cellular orextracellular macromolecules, such as proteins. For the purposes of thisdiscussion, such cellular and extracellular macromolecules are referredto herein as “binding partners.” Compounds that disrupt suchinteractions are useful for regulating the activity of the targetprotein. Such compounds can include, but are not limited, to moleculessuch as antibodies, peptides, and small molecules. In general, targetproteins for use in identifying agents that disrupt interactions are thetarget proteins identified herein. In alternative embodiments, theinvention provides methods for determining the ability of the testcompound to modulate the activity of a target protein through modulationof the activity of a downstream effector of a target protein. Forexample, the activity of the effector molecule on an appropriate targetcan be determined, or the binding of the effector to an appropriatetarget can be determined, as described herein.

To identify compounds that interfere with the interaction between thetarget protein and its binding partner(s), a reaction mixture containingthe target protein and the binding partner is prepared, under conditionsand for a time sufficient, to allow the two products to form a complex.To test an inhibitory agent, the reaction mixture is provided in thepresence (test sample) and absence (control sample) of the testcompound. The test compound can be initially included in the reactionmixture, or can be added at a time subsequent to the addition of thetarget gene and its cellular or extracellular binding partner. Controlreaction mixtures are incubated without the test compound or with acontrol compound. The formation of complexes between the target proteinand the cellular or extracellular binding partner is then detected. Theformation of a complex in the control reaction, and less formation ofcomplex in the reaction mixture containing the test compound, indicatesthat the compound interferes with the interaction of the target proteinand the interactive binding partner. Such compounds are candidatecompounds for inhibiting the expression or activity or a target protein.Additionally, complex formation within reaction mixtures containing thetest compound and normal target protein can also be compared to complexformation within reaction mixtures containing the test compound andmutant target gene product. This comparison can be important in thosecases wherein it is desirable to identify compounds that disruptinteractions of mutant but not normal target protein.

Binding assays can be carried out in a liquid phase or in heterogenousformats. In one type of heterogeneous assay system, either the targetprotein or the interactive cellular or extracellular binding partner, isanchored onto a solid surface (e.g., a microtiter plate), while thenon-anchored species is labeled, either directly or indirectly. Theanchored species can be immobilized by non-covalent or covalentattachments. Alternatively, an immobilized antibody specific for thespecies to be anchored can be used to anchor the species to the solidsurface.

To conduct the assay, the partner of the immobilized species is exposedto the coated surface with or without the test compound. After thereaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. Where the non-immobilized species is pre-labeled, the detectionof label immobilized on the surface indicates that complexes wereformed. Where the non-immobilized species is not pre-labeled, anindirect label can be used to detect complexes anchored on the surface;e.g., using a labeled antibody specific for the initiallynon-immobilized species (the antibody, in turn, can be directly labeledor indirectly labeled with, e.g., a labeled anti-Ig antibody). Dependingupon the order of addition of reaction components, test compounds thatinhibit complex formation or that disrupt preformed complexes can bedetected.

In another embodiment, modulators of target expression (RNA or protein)are identified. For example, a cell or cell-free mixture is contactedwith a test compound and the expression of target mRNA or proteinevaluated relative to the level of expression of target mRNA or proteinin the absence of the test compound. When expression of target mRNA orprotein is greater in the presence of the test compound than in itsabsence, the test compound is identified as a stimulator (candidatecompound) of target mRNA or protein expression. Alternatively, whenexpression of target mRNA or protein is less (statisticallysignificantly less) in the presence of the test compound than in itsabsence, the test compound is identified as an inhibitor (candidatecompound) of target mRNA or protein expression. The level of target mRNAor protein expression can be determined by methods described herein andmethods known in the art such as Northern blot or Western blot fordetecting target mRNA or protein, respectively.

In another aspect, the methods described herein pertain to a combinationof two or more of the assays described herein. For example, a modulatingagent can be identified using a cell-based or a cell-free assay, and theability of the agent to modulate the activity of a target protein can beconfirmed in vivo, e.g., in an animal such as an animal model forParkinson's disease.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent (compound) identified asdescribed herein (e.g., a target protein modulating agent, an anti sensenucleic acid molecule, an siRNA, a target protein-specific antibody, ora target protein-binding partner) in an appropriate animal model todetermine the efficacy, toxicity, side effects, or mechanism of action,of treatment with such an agent. Furthermore, novel agents identified bythe above-described screening assays can be used for treatments asdescribed herein.

Compounds that modulate target protein expression or activity (targetprotein modulators) can be tested for their ability to affect metaboliceffects associated with the target protein, e.g., with decreasedexpression or activity of target protein using methods known in the artand methods described herein. For example, the ability of a compound tomodulate alpha-synuclein mediated toxicity can be tested using an invitro or in vivo model for Parkinson's disease.

Target Protein Modulators

Methods of modulating target protein expression or activity can beaccomplished using a variety of compounds including nucleic acidmolecules that are targeted to a target nucleic acid sequence orfragment thereof, or to a target protein. Compounds that may be usefulfor inhibiting target protein expression or activity includepolynucleotides, polypeptides, small non-nucleic acid organic molecules,small inorganic molecules, antibodies or fragments thereof, antisenseoligonucleotides, siRNAs, and ribozymes. Methods of identifying suchcompounds are described herein.

RNA Inhibition (RNAi)

Molecules that are targeted to a target RNA are useful for the methodsdescribed herein, e.g., inhibition of target protein expression, e.g.,for treating a synucleinopathy such as Parkinson's disease. Examples ofnucleic acids include siRNAs. Other such molecules that function usingthe mechanisms associated with RNAi can also be used includingchemically modified siRNAs and vector driven expression of hairpin RNAthat are then cleaved to siRNA. The nucleic acid molecules or constructsthat are useful as described herein include dsRNA (e.g., siRNA)molecules comprising 16-30, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of thestrands is substantially identical, e.g., at least 80% (or more, e.g.,85%, 90%, 95%, or 100%) identical, e.g., having 3, 2, 1, or 0 mismatchednucleotide(s), to a target region in the mRNA, and the other strand iscomplementary to the first strand. The dsRNA molecules can be chemicallysynthesized, can transcribed be in vitro from a DNA template, or can betranscribed in vivo from, e.g., shRNA. The dsRNA molecules can bedesigned using methods known in the art, e.g., Dharmacon.com (see,siDESIGN CENTER) or “The siRNA User Guide,” available on the Internet atmpibpc.gwdg.de/abteilunge-n/100/105/sirna.html.

Negative control siRNAs (“scrambled”) generally have the same nucleotidecomposition as the selected siRNA, but without significant sequencecomplementarity to the appropriate genome. Such negative controls can bedesigned by randomly scrambling the nucleotide sequence of the selectedsiRNA; a homology search can be performed to ensure that the negativecontrol lacks homology to any other gene in the appropriate genome.Controls can also be designed by introducing an appropriate number ofbase mismatches into the selected siRNA sequence.

The nucleic acid compositions that are useful for the methods describedherein include both siRNA and crosslinked siRNA derivatives.Crosslinking can be used to alter the pharmacokinetics of thecomposition, for example, to increase half-life in the body. Thus, theinvention includes siRNA derivatives that include siRNA having twocomplementary strands of nucleic acid, such that the two strands arecrosslinked. For example, a 3′ OH terminus of one of the strands can bemodified, or the two strands can be crosslinked and modified at the 3′OHterminus. The siRNA derivative can contain a single crosslink (e.g., apsoralen crosslink). In some cases, the siRNA derivative has at its 3′terminus a biotin molecule (e.g., a photocleavable biotin), a peptide(e.g., a Tat peptide), a nanoparticle, a peptidomimetic, organiccompounds (e.g., a dye such as a fluorescent dye), or dendrimer.Modifying SiRNA derivatives in this way can improve cellular uptake orenhance cellular targeting activities of the resulting siRNA derivativeas compared to the corresponding siRNA, are useful for tracing the siRNAderivative in the cell, or improve the stability of the siRNA derivativecompared to the corresponding siRNA.

The nucleic acid compositions described herein can be unconjugated orcan be conjugated to another moiety, such as a nanoparticle, to enhancea property of the compositions, e.g., a pharmacokinetic parameter suchas absorption, efficacy, bioavailability, and/or half-life. Theconjugation can be accomplished using methods known in the art, e.g.,using the methods of Lambert et al., Drug Deliv. Rev., 47, 99-112, 2001(describes nucleic acids loaded to polyalkylcyanoacrylate (PACA)nanoparticles); Fattal et al., J. Control Release, 53:137-143, 1998(describes nucleic acids bound to nanoparticles); Schwab et al., Ann.Oncol., 5 Suppl. 4:55-8, 1994 (describes nucleic acids linked tointercalating agents, hydrophobic groups, polycations or PACAnanoparticles); and Godard et al., Eur. J. Biochem., 232:404-410, 1995(describes nucleic acids linked to nanoparticles).

The nucleic acid molecules can also be labeled using any method known inthe art; for instance, the nucleic acid compositions can be labeled witha fluorophore, e.g., Cy3, fluorescein, or rhodamine. The labeling can becarried out using a kit, e.g., the SILENCER™ siRNA labeling kit(Ambion). Additionally, the molecule can be radiolabeled, e.g., using³H, ³²P, or other appropriate isotope. Synthetic siRNAs can be deliveredinto cells by cationic liposome transfection and electroporation.Sequences that are modified to improve their stability can be used. Suchmodifications can be made using methods known in the art (e.g.,siSTABLE™, Dharmacon). Such stabilized molecules are particularly usefulfor in vivo methods such as for administration to a subject to decreasetarget protein expression. Longer term expression can also be achievedby delivering a vector that expresses the siRNA molecule (or othernucleic acid) to a cell, e.g., a fat, liver, or muscle cell. Severalmethods for expressing siRNA duplexes within cells from recombinant DNAconstructs allow longer-term target gene suppression in cells, includingmammalian Pol III promoter systems (e.g., HI or U6/snRNA promotersystems (Tuschl, Nature Biotechnol., 20:440-448, 2002) capable ofexpressing functional double-stranded siRNAs; (Bagella et al., J. Cell.Physiol., 177:206-1998; Lee et al., Nature Biotechnol., 20:500-505,2002; Paul et al., Nature Biotechnol., 20:505-508, 2002; Yu et al.,Proc. Natl. Acad. Sci. USA, 99(9):6047-6052, 2002; Sui et al., Proc.Natl. Acad. Sci. USA, 99(6):5515-5520, 2002). Transcriptionaltermination by RNA Pol III occurs at runs of four consecutive T residuesin the DNA template, providing a mechanism to end the siRNA transcriptat a specific sequence. The siRNA is complementary to the sequence ofthe target gene in 5′-3′ and 3′-5′ orientations, and the two strands ofthe siRNA can be expressed in the same construct or in separateconstructs. Hairpin siRNAs, driven by H1 or U6 snRNA promoter andexpressed in cells, can inhibit target gene expression (Bagella et al.,1998, supra; Lee et al., 2002, supra; Paul et al., 2002, supra; Yu etal., 2002, supra; Sui et al., 2002, supra). Constructs containing siRNAsequence under the control of T7 promoter also make functional siRNAswhen cotransfected into the cells with a vector expression T7 RNApolymerase (Jacque, Nature, 418:435-438, 2002).

Animal cells express a range of noncoding RNAs of approximately 22nucleotides termed micro RNA (miRNAs) and can regulate gene expressionat the post transcriptional or translational level during animaldevelopment. miRNAs are excised from an approximately 70 nucleotideprecursor RNA stem-loop. By substituting the stem sequences of the miRNAprecursor with miRNA sequence complementary to the target mRNA, a vectorconstruct that expresses the novel miRNA can be used to produce siRNAsto initiate RNAi against specific mRNA targets in mammalian cells (Zeng,Mol. Cell, 9:1327-1333, 2002). When expressed by DNA vectors containingpolymerase III promoters, micro-RNA designed hairpins can silence geneexpression (McManus, RNA 8:842-850, 2002). Viral-mediated deliverymechanisms can also be used to induce specific silencing of targetedgenes through expression of siRNA, for example, by generatingrecombinant adenoviruses harboring siRNA under RNA Pol II promotertranscription control (Xia et al., Nat Biotechnol., 20(10): 1006-10,2002).

Injection of the recombinant adenovirus vectors into transgenic miceexpressing the target genes of the siRNA results in in vivo reduction oftarget gene expression. In an animal model, whole-embryo electroporationcan efficiently deliver synthetic siRNA into post-implantation mouseembryos (Calegari et al., Proc. Natl. Acad. Sci. USA, 99:14236-14240,2002). In adult mice, efficient delivery of siRNA can be accomplished by“high-pressure” delivery technique, a rapid injection (within 5 seconds)of a large volume of siRNA containing solution into animal via the tailvein (Liu, Gene Ther., 6:1258-1266, 1999; McCaffrey, Nature, 418:38-39,2002; Lewis, Nature Genetics, 32:107-108, 2002). Nanoparticles andliposomes can also be used to deliver siRNA into animals. Likewise, insome embodiments, viral gene delivery, direct injection, nanoparticleparticle-mediated injection, or liposome injection may be used toexpress siRNA in humans.

In some cases, a pool of siRNAs is used to modulate the expression of atarget gene. The pool is composed of at least 2, 3, 4, 5, 8, or 10different sequences targeted to the target gene.

SiRNAs or other compositions that inhibit target protein expression oractivity are effective for ameliorating undesirable effects of adisorder related to alpha synuclein toxicity when target RNA levels arereduced by at least 25%, 50%, 75%, 90%, or 95%. In some cases, it isdesired that target RNA levels be reduced by not more than 10%, 25%,50%, or 75%. Methods of determining the level of target gene expressioncan be determined using methods known in the art. For example, the levelof target RNA can be determined using Northern blot detection on asample from a cell line or a subject. Levels of target protein can alsobe measured using, e.g., an immunoassay method.

Antisense Nucleic Acids

Antisense nucleic acids are useful for inhibiting a target protein. Suchantisense nucleic acid molecules, i.e., nucleic acid molecules whosenucleotide sequence is complementary to all or part of an mRNA encodinga target protein. An antisense nucleic acid molecule can be antisense toall or part of a non-coding region of the coding strand of a nucleotidesequence encoding a target protein. The non-coding regions (“5′ and 3′untranslated regions”) are the 5′ and 3′ sequences that flank the codingregion and are not translated into amino acids.

Based upon the nucleotide sequences disclosed herein, one of skill inthe art can easily choose and synthesize any of a number of appropriateantisense molecules to target a gene described herein. For example, a“gene walk” comprising a series of oligonucleotides of 15-30 nucleotidesspanning the length of a nucleic acid (e.g., a target nucleic acid) canbe prepared, followed by testing for inhibition of expression of thegene. Optionally, gaps of 5-10 nucleotides can be left between theoligonucleotides to reduce the number of oligonucleotides synthesizedand tested.

An antisense oligonucleotide can be, for example, about 5, 10, 15, 20,25, 30, 35, 40, 45, or 50 nucleotides or more in length. An antisensenucleic acid described herein can be constructed using chemicalsynthesis and enzymatic ligation reactions using procedures known in theart. For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, e.g., phosphorothioate derivatives and acridine substitutednucleotides can be used. Examples of modified nucleotides which can beused to generate the antisense nucleic acid include 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridin-e,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methyl cytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiour-acil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-β-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The new antisense nucleic acid molecules can be administered to amammal, e.g., a human patient. Alternatively, they can be generated insitu such that they hybridize with or bind to cellular mRNA and/orgenomic DNA encoding a selected polypeptide to thereby inhibitexpression, e.g., by inhibiting transcription and/or translation. Thehybridization can be by conventional nucleotide complementarities toform a stable duplex, or, for example, in the case of an antisensenucleic acid molecule which binds to DNA duplexes, through specificinteractions in the major groove of the double helix. An example of aroute of administration of antisense nucleic acid molecules of theinvention includes direct injection at a tissue site. Alternatively,antisense nucleic acid molecules can be modified to target selectedcells and then administered systemically. For example, for systemicadministration, antisense molecules can be modified such that theyspecifically bind to receptors or antigens expressed on a selected cellsurface, e.g., by linking the antisense nucleic acid molecules topeptides or antibodies that bind to cell surface receptors or antigens.The antisense nucleic acid molecules can also be delivered to cellsusing the vectors described herein. For example, to achieve sufficientintracellular concentrations of the antisense molecules, vectorconstructs can be used in which the antisense nucleic acid molecule isplaced under the control of a strong pol II or pol III promoter.

An antisense nucleic acid molecule can be an alpha-anomeric nucleic acidmolecule. An alpha-anomeric nucleic acid molecule forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual, beta-units, the strands run parallel to each other (Gaultier etal., Nucleic Acids Res., 15:6625-6641, 1987). The antisense nucleic acidmolecule can also comprise a 2′-o-methylribonucleotide (Inoue et al.,Nucleic Acids Res., 15:6131-6148, 1987) or a chimeric RNA-DNA analog(Inoue et al., FEBS Lett., 215:327-330, 1987).

Antisense molecules that are complementary to all or part of a targetgene described herein are also useful for assaying expression of suchgenes using hybridization methods known in the art. For example, theantisense molecule can be labeled (e.g., with a radioactive molecule)and an excess amount of the labeled antisense molecule is hybridized toan RNA sample. Unhybridized labeled antisense molecule is removed (e.g.,by washing) and the amount of hybridized antisense molecule measured.The amount of hybridized molecule is measured and used to calculate theamount of expression of the target gene. In general, antisense moleculesused for this purpose can hybridize to a sequence from a target geneunder high stringency conditions such as those described herein. Whenthe RNA sample is first used to synthesize cDNA, a sense molecule can beused. It is also possible to use a double-stranded molecule in suchassays as long as the double-stranded molecule is adequately denaturedprior to hybridization.

Ribozymes

Ribozymes that have specificity for a target nucleic acid sequence canalso be used to inhibit target gene expression. Ribozymes are catalyticRNA molecules with ribonuclease activity that are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region. Thus, ribozymes (e.g., hammerhead ribozymes(described in Haselhoff and Gerlach, Nature, 334:585-591, 1988)) can beused to catalytically cleave mRNA transcripts to thereby inhibittranslation of the protein encoded by the mRNA. Methods of designing andproducing ribozymes are known in the art (see, e.g., Scanlon, 1999,Therapeutic Applications of Ribozymes, Humana Press). A ribozyme havingspecificity for a target nucleic acid molecule or fragment thereof canbe designed based upon the nucleotide sequence of a target cDNA. Forexample, a derivative of a Tetrahymena L-19 IVS RNA can be constructedin which the nucleotide sequence of the active site is complementary tothe nucleotide sequence to be cleaved in a target RNA (Cech et al. U.S.Pat. No. 4,987,071; and Cech et al., U.S. Pat. No. 5,116,742).Alternatively, an mRNA encoding a target protein or fragment thereof canbe used to select a catalytic RNA having a specific ribonucleaseactivity from a pool of RNA molecules (See, e.g., Bartel and Szostak,Science, 261:1411-1418, 1993).

Nucleic acid molecules that form triple helical structures can also beused to modulate target protein expression. For example, expression of atarget protein can be inhibited by targeting nucleotide sequencescomplementary to the regulatory region of the gene encoding thepolypeptide (e.g., the promoter and/or enhancer) to form triple helicalstructures that prevent transcription of the gene in target cells. Seegenerally Helene, Anticancer Drug Des., 6(6):569-84, 1991; Helene, Ann.N. Y Acad. Sci., 660:27-36, 1992; and Maher, Bioassays, 14(12):807-15,1992.

A nucleic acid molecule for use as described herein can be modified atthe base moiety, sugar moiety or phosphate backbone to improve, e.g.,the stability, hybridization, or solubility of the molecule. Forexample, the deoxyribose phosphate backbone of a nucleic acid can bemodified to generate peptide nucleic acids (see Hyrup et al., Bioorganic& Medicinal Chem., 4(1): 5-23, 1996). Peptide nucleic acids (PNAs) arenucleic acid mimics, e.g., DNA mimics, in which the deoxyribosephosphate backbone is replaced by a pseudopeptide backbone and only thefour natural nucleobases are retained. The neutral backbone of PNAsallows for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid phase peptide synthesis protocols, e.g., as described inHyrup et al., 1996, supra; Perry-O′Keefe et al., Proc. Natl. Acad. Sci.USA, 93: 14670-675, 1996.

PNAs can be used in therapeutic and diagnostic applications. Forexample, PNAs can be used as antisense or antigene agents forsequence-specific modulation of gene expression by, e.g., inducingtranscription or translation arrest or inhibiting replication. PNAs canalso be used, e.g., in the analysis of single base pair mutations in agene by, e.g., PNA directed PCR clamping; as artificial restrictionenzymes when used in combination with other enzymes, e.g., S1 nucleases(Hyrup, 1996, supra; or as probes or primers for DNA sequence andhybridization (Hyrup, 1996, supra; Perry-O′Keefe et al., Proc. Natl.Acad. Sci. USA, 93: 14670-675, 1996).

PNAs can be modified, e.g., to enhance their stability or cellularuptake, by attaching lipophilic or other helper groups to PNA, by theformation of PNA-DNA chimeras, or by the use of liposomes or othertechniques of drug delivery known in the art. For example, PNA-DNAchimeras can be generated which may combine the advantageous propertiesof PNA and DNA.

Such chimeras allow DNA recognition enzymes, e.g., RNAse H and DNApolymerases, to interact with the DNA portion while the PNA portionwould provide high binding affinity and specificity. PNA-DNA chimerascan be linked using linkers of appropriate lengths selected in terms ofbase stacking, number of bonds between the nucleobases, and orientation(Hyrup,1996, supra). The synthesis of PNA-DNA chimeras can be performedas described in Hyrup,1996, supra, and Finn et al., Nucleic Acids Res.,24:3357-63, 1996. For example, a DNA chain can be synthesized on a solidsupport using standard phosphoramidite coupling chemistry and modifiednucleoside analogs. Compounds such as5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite can be usedas a link between the PNA and the 5′ end of DNA (Mag et al., NucleicAcids Res., 17:5973-88, 1989). PNA monomers are then coupled in astepwise manner to produce a chimeric molecule with a 5′ PNA segment anda 3′ DNA segment (Finn et al., Nucleic Acids Res., 24:3357-63, 1996).Alternatively, chimeric molecules can be synthesized with a 5′ DNAsegment and a 3′ PNA segment (Peterser et al., Bioorganic Med. Chem.Lett., 5:1119-11124, 1975).

A nucleic acid targeting a target nucleic acid sequence can includeappended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. USA,86:6553-6556, 1989; Lemaitre et al., Proc. Natl. Acad. Sci. USA,84:648-652, 1989; WO 88/09810) or the blood-brain barrier (see, e.g., WO89/10134). In addition, oligonucleotides can be modified withhybridization-triggered cleavage agents (see, e.g., Krol et al.,Bio/Techniques, 6:958-976, 1988) or intercalating agents (see, e.g.,Zon, Pharm. Res., 5:539-549, 1988). To this end, the oligonucleotide maybe conjugated to another molecule, e.g., a peptide, hybridizationtriggered cross-linking agent, transport agent, or ahybridization-triggered cleavage agent.

Polypeptides

Isolated target proteins, fragments thereof, and variants thereof areprovided herein. These polypeptides can be used, e.g., as immunogens toraise antibodies, in screening methods, or in methods of treatingsubjects, e.g., by administration of the target proteins. An “isolated”or “purified” polypeptide or biologically active portion thereof issubstantially free of cellular material or other contaminating proteinsfrom the cell or tissue source from which the protein is derived, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. The language “substantially free of cellularmaterial” includes preparations of polypeptides in which the polypeptideof interest is separated from cellular components of the cells fromwhich it is isolated or recombinantly produced. Thus, a polypeptide thatis substantially free of cellular material includes preparations ofpolypeptides having less than about 30%, 20%, 10%, or 5% (by dry weight)of heterologous protein (also referred to herein as “contaminatingprotein”). In general, when the polypeptide or biologically activeportion thereof is recombinantly produced, it is also substantially freeof culture medium, i.e., culture medium represents less than about 20%,10%, or 5% of the volume of the protein preparation. In general, whenthe polypeptide is produced by chemical synthesis, it is substantiallyfree of chemical precursors or other chemicals, i.e., it is separatedfrom chemical precursors or other chemicals that are involved in thesynthesis of the polypeptide. Accordingly such preparations of thepolypeptide have less than about 30%, 20%, 10%, or 5% (by dry weight) ofchemical precursors or compounds other than the polypeptide of interest.

Expression of target proteins can be assayed to determine the amount ofexpression. Methods for assaying protein expression are known in the artand include Western blot, immunoprecipitation, and radioimmunoassay.

As used herein, a “biologically active portion” of a target proteinincludes a fragment of a target protein that participates in aninteraction between a target proteins and a non-target protein.Biologically active portions of a target protein include peptidesincluding amino acid sequences sufficiently homologous to the amino acidsequence of a target protein that includes fewer amino acids than afull-length target protein, and exhibits at least one activity of atarget protein. Typically, biologically active portions include a domainor motif with at least one activity of the target protein. Abiologically active portion of a target protein can be a polypeptidethat is, for example, 10, 25, 50, 100, 200 or more amino acids inlength. Biologically active portions of a target protein can be used astargets for developing agents that modulate a target protein mediatedactivity, e.g., compounds that inhibit target protein activity.

In some embodiments, the target protein has a sequence identical to asequence disclosed herein (e.g., an amino acid sequence found under aGenBankTM Accession Number listed in Table 1). Other useful polypeptidesare substantially identical (e.g., at least about 45%, 55%, 65%, 75%,85%, 95%, or 99% identical) to a sequence disclosed herein (e.g., anamino acid sequence found under a GenBankTM Accession Number listed inTable 1) and (a) retains the functional activity of the target proteinyet differs in amino acid sequence due to natural allelic variation ormutagenesis, or (b) exhibits an altered functional activity (e.g., as adominant negative) where desired. Provided herein are variants that havean altered amino acid sequence which can function as either agonists(mimetics) or as antagonists. Variants can be generated by mutagenesis,e.g., discrete point mutation or truncation. An agonist can retainsubstantially the same, or a subset, of the biological activities of thenaturally occurring form of the polypeptide. An antagonist of apolypeptide can inhibit one or more of the activities of the naturallyoccurring form of the polypeptide by, for example, competitively bindingto a downstream or upstream member of a cellular signaling cascade thatincludes the polypeptide. Thus, specific biological effects can beelicited by treatment with a variant of limited function. Treatment of asubject with a variant having a subset of the biological activities ofthe naturally occurring form of the polypeptide can have fewer sideeffects in a subject relative to treatment with the naturally occurringform of the polypeptide. In some embodiments, the variant target proteinis a dominant negative form of the target protein. Dominant negativesare desired, e.g., in methods in which inhibition of target proteinaction is desired.

Also provided herein are chimeric or fusion proteins.

The comparison of sequences and determination of percent identitybetween two sequences is accomplished using a mathematical algorithm.The percent identity between two amino acid sequences is determinedusing the Needleman and Wunsch, J. Mol. Biol., 48:444-453, 1970)algorithm, which has been incorporated into the GAP program in the GCGsoftware package (available on the Internet at gcg.com), using either aBlossum 62 matrix or a PAM250 matrix, and a gap weight of 16 and alength weight of 1. The percent identity between two nucleotidesequences is determined using the GAP program in the GCG softwarepackage (also available on the Internet at gcg.com), using aNWSgapdna.CMP matrix, a gap weight of 40, and a length weight of 1.

In general, percent identity between amino acid sequences referred toherein is determined using the BLAST 2.0 program, which is available tothe public on the Internet at ncbi.nlm.nih.gov/BLAST. Sequencecomparison is performed using an ungapped alignment and using thedefault parameters (Blossum 62 matrix, gap existence cost of 11, perresidue gap cost of 1, and a lambda ratio of 0.85). The mathematicalalgorithm used in BLAST programs is described in Altschul et al.,Nucleic Acids Research 25:3389-3402, 1997.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a target protein isgenerally replaced with another amino acid residue from the same sidechain family. Alternatively, mutations can be introduced randomly alongall or part of a target protein coding sequence, such as by saturationmutagenesis, and the resultant mutants can be screened for targetprotein biological activity to identify mutants that retain activity.The encoded protein can be expressed recombinantly and the activity ofthe protein can be determined.

Antibodies

A target protein, or a fragment thereof, can be used as an immunogen togenerate antibodies using standard techniques for polyclonal andmonoclonal antibody preparation. The full-length polypeptide or proteincan be used or, alternatively, antigenic peptide fragments can be usedas immunogens. The antigenic peptide of a protein comprises at least 8(e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acidsequence of a target protein, and encompasses an epitope of a targetprotein such that an antibody raised against the peptide forms aspecific immune complex with the polypeptide.

An immunogen typically is used to prepare antibodies by immunizing asuitable subject (e.g., rabbit, goat, mouse or other mammal). Anappropriate immunogenic preparation can contain, for example, arecombinantly expressed or a chemically synthesized polypeptide. Thepreparation can further include an adjuvant, such as Freund's completeor incomplete adjuvant, or similar immunostimulatory agent.

Polyclonal antibodies can be prepared as described above by immunizing asuitable subject with a target protein as an immunogen. The antibodytiter in the immunized subject can be monitored over time by standardtechniques, such as with an enzyme linked immunosorbent assay (ELISA)using immobilized polypeptide. If desired, the antibody molecules can beisolated from the mammal (e.g., from the blood) and further purified bywell-known techniques, such as protein A chromatography to obtain theIgG fraction. At an appropriate time after immunization, e.g., when thespecific antibody titers are highest, antibody-producing cells can beobtained from the subject and used to prepare monoclonal antibodies bystandard techniques, such as the hybridoma technique originallydescribed by Kohler and Milstein, Nature, 256:495-497, 1975, the human Bcell hybridoma technique (Kozbor et al., Immunol. Today, 4:72, 1983),the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985) or triomatechniques. The technology for producing hybridomas is well known (seegenerally Current Protocols in Immunology, 30 1994, Coligan et al.(eds.) John Wiley & Sons, Inc., New York, N.Y.). Hybridoma cellsproducing a monoclonal antibody are detected by screening the hybridomaculture supernatants for antibodies that bind the polypeptide ofinterest, e.g., using a standard ELISA assay.

As an alternative to preparing monoclonal antibody-secreting hybridomas,a monoclonal antibody directed against a polypeptide can be identifiedand isolated by screening a recombinant combinatorial immunoglobulinlibrary (e.g., an antibody phage display library) with the polypeptideof interest. Kits for generating and screening phage display librariesare commercially available (e.g., the Pharmacia Recombinant PhageAntibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP™Phage Display Kit, Catalog No. 240612). Additionally, examples ofmethods and reagents particularly amenable for use in generating andscreening antibody display library can be found in, for example, U.S.Pat. No. 5,223,409; WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679;WO 93/01288; WO 92/01047; WO 92/09690; WO 90/02809; Fuchs et al.,Bio/Technology, 9:1370-1372, 1991; Hay et al., Hum. Antibod. Hybridomas,3:81-85, 1992; Huse et al., Science, 246:1275-1281, 1989; Griffiths etal., EMBO J., 12:725-734, 1993.

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, including both human and non-human portions,which can be made using standard recombinant DNA techniques, areprovided herein. Such chimeric and humanized monoclonal antibodies canbe produced by recombinant DNA techniques known in the art, for exampleusing methods described in WO 87/02671; European Patent Application184,187; European Patent Application 171,496; European PatentApplication 173,494; WO 86/01533; U.S. Pat. No. 4,816,567; EuropeanPatent Application 125,023; Better et al., Science, 240:1041-1043, 1988;Liu et al., Proc. Natl. Acad. Sci. USA 84:3439-3443, 1987; Liu et al.,J. Immunol., 139:3521-3526, 1987; Sun et al., Proc. Natl. Acad. Sci.USA, 84:214-218, 1987; Nishimura et al., Canc. Res., 47:999-1005, 1987;Wood et al., Nature, 314:446-449, 1985; and Shaw et al., J. Natl. CancerInst., 80:1553-1559, 1988); Morrison, Science, 229:1202-1207, 1985; Oiet al., Bio/Techniques, 4:214, 1986; U.S. Pat. No. 5,225,539; Jones etal., Nature, 321:552-525, 1986; Verhoeyan et al., Science, 239:1534,1988; and Beidler et al., J. Immunol., 141:4053-4060, 1988.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Such antibodies can be produced usingtransgenic mice which are incapable of expressing endogenousimmunoglobulin heavy and light chains genes, but which can express humanheavy and light chain genes. The transgenic mice are immunized in thenormal fashion with a selected antigen, e.g., all or a portion of atarget protein. Monoclonal antibodies directed against the antigen canbe obtained using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG IgA, and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar (Int. Rev. Immunol., 13:65-93, 1995). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., U.S. Pat. Nos. 5,625,126; 5,633,425; 5,569,825; 5,661,016; and5,545,806.

Completely human antibodies that recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a murineantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Biotechnology,12:899-903, 1994).

An antibody directed against a target protein can be used to detect thepolypeptide (e.g., in a cellular lysate or cell supernatant) to evaluateits abundance and pattern of expression. The antibodies can also be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g., for example, to determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling theantibody to a detectable substance. Examples of detectable substancesinclude various enzymes, prosthetic groups, fluorescent materials,luminescent materials, bioluminescent materials, and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;examples of suitable prosthetic group complexes includestreptavidin/biotin and avidin/biotin; examples of suitable fluorescentmaterials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Pharmaceutical Compositions

A test compound that has been screened by a method described herein anddetermined to modulate target protein expression or activity, can beconsidered a candidate compound. A candidate compound that has beenscreened, e.g., in an in vivo model of a synucleinopathy such asParkinson's disease, and determined to have a desirable effect on thedisorder, can be considered a candidate therapeutic agent. Candidatetherapeutic agents, once screened in a clinical setting, are therapeuticagents. Candidate therapeutic agents and therapeutic agents can beoptionally optimized and/or derivatized, and formulated withphysiologically acceptable excipients to form pharmaceuticalcompositions.

The compounds described herein that can modulate target proteinexpression or activity can be incorporated into pharmaceuticalcompositions. Such compositions typically include the compound and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” includes solvents, dispersionmedia, coatings, antibacterial and antifungal agents, isotonic andabsorption delaying agents, and the like, compatible with pharmaceuticaladministration. Supplementary active compounds can also be incorporatedinto the compositions.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude parenteral, e.g., intravenous, intradermal, subcutaneous, oral(e.g., inhalation), transdermal (topical), transmucosal, and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfate; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. A parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be desirable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the methods of preparation can includevacuum drying or freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. Dosage units can also be accompanied byinstructions for use.

Toxicity and therapeutic efficacy of such compounds can be determinedknown pharmaceutical procedures in cell cultures or experimental animals(animal models of synucleinopathies, e.g., Parkinson's disease). Theseprocedures can be used, e.g., for determining the LD50 (the dose lethalto 50% of the population) and the ED50 (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio LD50/ED50. Compounds that exhibit high therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in to minimize potential damageto uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies generally within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For a compound usedas described herein (e.g., for treating a synucleinopathy in a subject),the therapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range that includes the IC50 (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

As defined herein, a therapeutically effective amount of protein orpolypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, about 0.01 to 25 mg/kg body weight, about 0.1 to 20mg/kg body weight, about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to7 mg/kg, or 5 to 6 mg/kg body weight. The protein or polypeptide can beadministered one time per week for between about 1 to 10 weeks,generally between 2 to 8 weeks, between about 3 to 7 weeks, or for about4, 5, or 6 weeks. One in the art will appreciate that certain factorsmay influence the dosage and timing required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of a protein, polypeptide, orantibody can include a single treatment or can include a series oftreatments.

For antibodies or a fragment thereof, the dosage is about 0.1 mg/kg ofbody weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to actin the brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate.Generally, partially human antibodies and fully human antibodies have alonger half-life within the human body than other antibodies.Accordingly, lower dosages and less frequent administration is oftenpossible with such species-matched antibodies. Modifications such aslipidation can be used to stabilize antibodies and to enhance uptake andtissue penetration (e.g., into the brain). A method for lipidation ofantibodies is described by Cruikshank et al. (J. Acquired ImmuneDeficiency Syndromes and Human Retrovirology, 14:193, 1997).

Compounds that modulate expression or activity of a target protein aredescribed herein. Such a compound can be a small molecule. For example,such small molecules include, but are not limited to, peptides,peptidomimetics (e.g., peptoids), amino acids, amino acid analogs,polynucleotides, polynucleotide analogs, nucleotides, nucleotideanalogs, organic or inorganic compounds (i.e., including heteroorganicand organometallic compounds) having a molecular weight less than about10,000 grams per mole, organic or inorganic compounds having a molecularweight less than about 5,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 1,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 500 grams per mole, and salts, esters, and other pharmaceuticallyacceptable forms of such compounds.

Examples of doses include milligram or microgram amounts of the smallmolecule per kilogram of subject or sample weight (e.g., about 1microgram per kilogram to about 500 milligrams per kilogram, about 100micrograms per kilogram to about 5 milligrams per kilogram, or about 1microgram per kilogram to about 50 micrograms per kilogram). It isfurthermore understood that appropriate doses of a small molecule dependupon the potency of the small molecule with respect to the expression oractivity to be modulated. When one or more of these small molecules isto be administered to an animal (e.g., a human) to modulate expressionor activity of a polypeptide or nucleic acid of the invention, aphysician, veterinarian, or researcher may, for example, prescribe arelatively low dose at first, subsequently increasing the dose until anappropriate response is obtained. In addition, it is understood that thespecific dose level for any particular animal subject will depend upon avariety of factors including the activity of the specific compoundemployed, the age, body weight, general health, gender, and diet of thesubject, the time of administration, the route of administration, therate of excretion, any drug combination, and the degree of expression oractivity to be modulated.

An antibody (or fragment thereof) can be conjugated to a therapeuticmoiety such as a cytotoxin, a therapeutic agent, or a radioactive metalion. A cytotoxin or cytotoxic agent includes any agent that isdetrimental to cells. Examples include taxol, cytochalasin B, gramicidinD, ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

A nucleic acid molecule that is useful for modulating target proteinexpression or activity can be inserted into a vector and the resultingvector used as gene therapy vector. Gene therapy vectors can bedelivered to a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (Proc. Natl. Acad Sci. USA,91:3054-3057, 1994). The pharmaceutical preparation of the gene therapyvector can include the gene therapy vector in an acceptable diluent, orcan comprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Methods of Treatment

Compounds described herein and those identified as described herein canbe used to treat a subject that is at risk for or has a diseaseassociated with alpha-synuclein toxicity and/or the formation,deposition, accumulation, or persistence of synuclein fibrils, includingalpha-synuclein fibrils. In certain embodiments, diseases includesynucleinopathies such as Parkinson's disease (including Parkinson'sdisease chemically induced by exposure to environmental agents such aspesticides, insecticides, or herbicides and/or metals such as manganese,aluminum, cadmium, copper, or zinc, SNCA gene-linked Parkinson'sdisease, sporadic or idiopathic Parkinson's disease, or Parkin- orLRRK2-linked Parkinson's disease), dementia with Lewy bodies, pureautonomic failure, multiple system atrophy, incidental Lewy bodydisease, pantothenate kinase-associated neurodegeneration, Alzheimer'sdisease, Down's Syndrome, Gaucher disease, or the Parkinsonism-dementiacomplex of Guam.

Methods of identifying an individual at risk for or having asynucleinopathy are known in the art. Thus, methods and compositions forboth prophylactic and therapeutic methods of treating a subject at riskof (or susceptible to) a synucleinopathy are described herein. Forexample, an individual who is at risk of developing Parkinson's disease(e.g., an individual whose family history includes Parkinson's disease)and/or has signs he/she will develop Parkinson's disease can be treatedwith the compounds and methods described herein.

As used herein, the term “treatment” is defined as the application oradministration of a therapeutic compound to a patient, or application oradministration of a therapeutic compound to an isolated tissue or cellline from a patient, who has a disease, a symptom of disease or apredisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisease, the symptoms of disease or the predisposition toward disease. Atherapeutic compound includes, but is not limited to, small moleculessuch as small non-nucleic acid organic molecules, small inorganicmolecules, peptides, synthetic peptides, antibodies, natural nucleicacid molecules (such as ribozymes, siRNAs, and antisenseoligonucleotides), and molecules containing nucleic acid analogs.

Provided herein are methods for preventing in a subject (e.g., a human),a synucleinopathy, by administering to the subject a target protein or acompound that modulates target protein expression or at least one targetprotein activity. Subjects at risk for a disease that is caused orcontributed to by aberrant or unwanted target protein expression oractivity can be identified by, for example, any or a combination ofdiagnostic or prognostic assays as described herein. Administration of aprophylactic compound can occur prior to the manifestation of symptomscharacteristic of full-blown disease, such that the disease or disorderis prevented or, alternatively, delayed in its progression. Methodsknown in the art can be used to determine the efficacy of the treatment.The appropriate compound used for treating the subject can be determinedbased on screening assays described herein.

It is possible that some cases of synucleinopathies are caused, at leastin part, by an abnormal level of a target gene product, or by thepresence of a target protein exhibiting abnormal activity. As such, thereduction in the level and/or activity of such gene products will bringabout the amelioration of disorder symptoms.

As discussed, successful treatment of synucleinopathies can be broughtabout by techniques that serve to inhibit the expression or activity ofselected target gene products. For example, compounds, e.g., an agentidentified using one or more of the assays described above, that provesto exhibit negative modulatory activity, can be used as described hereinto prevent and/or ameliorate symptoms of synucleinopathies. Suchmolecules can include, but are not limited to, peptides,phosphopeptides, small organic or inorganic molecules, or antibodies(including, for example, polyclonal, monoclonal, humanized,anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab′)₂and Fab expression library fragments, scFV molecules, andepitope-binding fragments thereof).

Further, siRNA, antisense, and ribozyme molecules that inhibitexpression of a target gene can also be used in accordance with themethods described herein to reduce the level of target proteinexpression, thus effectively reducing the level of target proteinactivity. Triple helix molecules can be utilized to reduce the level oftarget protein activity.

Another method by which nucleic acid molecules can be utilized intreating or preventing a disease that can be treated by modulatingtarget protein expression is through the use of aptamer moleculesspecific for target protein. Aptamers are nucleic acid molecules havinga tertiary structure that permits them to specifically bind to proteinligands (e.g., Osbome, et al., Curr. Opin. Chem. Biol., 1: 5-9, 1997;and Patel, Curr. Opin. Chem. Biol., 1:32-46, 1997). Since nucleic acidmolecules may be more conveniently introduced into target cells thantherapeutic protein molecules may be, aptamers offer a method by whichtarget protein activity can be specifically decreased without theintroduction of drugs or other molecules that may have pluripotenteffects.

An antibody that specifically recognizes a target protein can also beused. Lipofectin ^(TM) or liposomes can be used to deliver the antibodyor a fragment of the Fab region that binds to the target protein in acell. Where fragments of the antibody are used, the smallest inhibitoryfragment that binds to the target antigen is generally used. Forexample, peptides having an amino acid sequence corresponding to the Fvregion of the antibody can be used. Alternatively, single chainneutralizing antibodies that bind to an intracellular target protein canalso be administered. Such single chain antibodies can be administered,for example, by expressing nucleotide sequences encoding single-chainantibodies within the target cell population (e.g., Marasco et al.,Proc. Natl. Acad. Sci. USA, 90:7889-7893, 1993).

The identified compounds that inhibit target gene expression, synthesisand/or activity can be administered to a patient at therapeuticallyeffective doses to prevent, treat, or ameliorate synucleinopathies. Atherapeutically effective dose refers to that amount of the compoundsufficient to result in amelioration of symptoms of the disorders.Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures as described above.

The dosage of such compounds lies generally within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage can vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used as described herein, the therapeutically effective dosecan be estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

Another example of determination of effective dose for an individual isthe ability to directly assay levels of “free” and “bound” compound inthe serum of the test subject. Such assays may utilize antibody mimicsand/or “biosensors” that have been created through molecular imprintingtechniques. The compound that is able to modulate target proteinactivity is used as a template, or “imprinting molecule,” to spatiallyorganize polymerizable monomers prior to their polymerization withcatalytic reagents. The subsequent removal of the imprinted moleculeleaves a polymer matrix that contains a repeated “negative image” of thecompound and is able to selectively rebind the molecule under biologicalassay conditions. A detailed review of this technique can be seen inAnsell et al., Current Opinion in Biotechnology, 7:89-94, 1996 and inShea (Trends in Polymer Science, 2:166-173, 1994). Such “imprinted”affinity matrixes are amenable to ligand-binding assays, whereby theimmobilized monoclonal antibody component is replaced by anappropriately imprinted matrix. An example of the use of such matrixesin this way can be seen in Vlatakis et al. (Nature, 361:645-647, 1993).Through the use of isotope-labeling, the “free” concentration ofcompound that modulates the expression or activity of a target proteincan be readily monitored and used in calculations of IC₅₀.

Such “imprinted” affinity matrixes can also be designed to includefluorescent groups whose photon-emitting properties measurably changeupon local and selective binding of target compound. These changes canbe readily assayed in real time using appropriate fiberoptic devices, inturn allowing the dose in a test subject to be quickly optimized basedon its individual IC₅₀. A rudimentary example of such a “biosensor” isdiscussed in Kriz et al., Analytical Chemistry, 67:2142-2144, 1995.

Target protein expression or activity can be modulated for therapeuticpurposes. Accordingly, in some embodiments, the modulatory methodsdescribed herein involve contacting a cell with a compound thatmodulates one or more of the activities of a target protein associatedwith the cell. A compound that modulates target protein activity can bea compound as described herein, such as a nucleic acid or a protein, anaturally-occurring target molecule of a target protein (e.g., a targetprotein substrate or receptor), a target protein antibody, a targetprotein agonist or antagonist, a peptidomimetic of a target proteinagonist or antagonist, or other small molecule.

In one embodiment, the compound stimulates one or more target proteinactivities. Examples of such stimulatory compounds include active targetprotein and a nucleic acid molecule encoding the target protein. Inanother embodiment, the compound inhibits one or more target proteinactivities. Examples of such inhibitory compounds include antisensetarget nucleic acid molecules, anti-target protein antibodies, andtarget protein inhibitors. These modulatory methods can be performed invitro (e.g., by culturing cells with the compound and returning thecells to a subject) or, alternatively, in vivo (e.g., by administeringthe compound to a subject). As such, the new methods include treating anindividual afflicted with a disease or disorder characterized byaberrant or unwanted expression or activity of a target protein ornucleic acid molecule. In one embodiment, the methods involveadministering a compound (e.g., a compound identified by a screeningassay described herein), or combination of compounds that modulate(e.g., up regulates or down regulates) target protein expression oractivity. In another embodiment, the methods involve administering atarget protein or nucleic acid molecule as therapy to compensate forreduced, aberrant, or unwanted target protein expression or activity.

Stimulation of target protein activity is desirable in situations inwhich target protein is abnormally downregulated and/or in whichincreased target protein activity is likely to have a beneficial effect.For example, stimulation of target protein activity is desirable insituations in which a target protein is downregulated and/or in whichincreased target protein activity is likely to have a beneficial effect.Likewise, inhibition of target protein activity is desirable insituations in which target protein is abnormally upregulated and/or inwhich decreased target protein activity is likely to have a beneficialeffect.

In certain embodiments, one or more compounds (e.g., compounds thatmodulate expression or activity of different genes or proteins) can beadministered, together (simultaneously) or at different times(sequentially). In addition, such compounds can be administered withanother type(s) of compound(s) for treating a synucleinopathy. Forexample, an identified compound may be administered together withLevodopa (L-DOPA) for treating Parkinson's disease and/or therapeuticagents such as donepezil hydrochloride (Aracept), rivastigmine tartrate(Exelon), tacrine hydrochloride (Cognex), and/or galantaminehydrobromide (Reminyl).

A compound or pharmaceutical composition thereof described herein can beadministered to a subject as a combination therapy with anothertreatment, e.g., a treatment for a synucleinopathy (see immediatelyabove). For example, the combination therapy can include administeringto the subject (e.g., a human patient) one or more additional agentsthat provide a therapeutic benefit to the subject who has, or is at riskof developing, (or suspected of having) a synucleinopathy such as any ofthose described herein. Thus, the compound or pharmaceutical compositionand the one or more additional agents can be administered at the sametime. Alternatively, the compound can be administered first in time andthe one or more additional agents administered second in time. The oneor more additional agents can be administered first in time and thecompound administered second in time. The compound can replace oraugment a previously or currently administered therapy. For example,upon treating with a compound of the invention, administration of theone or more additional agents can cease or diminish, e.g., beadministered at lower levels. Administration of the previous therapy canalso be maintained. In some instances, a previous therapy can bemaintained until the level of the compound (e.g., the dosage orschedule) reaches a level sufficient to provide a therapeutic effect.The two therapies can be administered in combination.

It will be appreciated that in instances where a previous therapy isparticularly toxic (e.g., a treatment for a synucleinopathy withsignificant side-effect profiles) or poorly tolerated by a subject(e.g., a patient), administration of a compound of the invention can beused to offset and/or lessen the amount of the previous therapy to alevel sufficient to give the same or improved therapeutic benefit, butwithout the toxicity.

In some instances, when the subject is administered a compound orpharmaceutical composition of the invention the first therapy is halted.The subject can be monitored for a first pre-selected result, e.g., animprovement in one or more symptoms of a synucleinopathy such as any ofthose described herein (e.g., see above). In some cases, where the firstpre-selected result is observed, treatment with the compound isdecreased or halted. The subject can then be monitored for a secondpre-selected result after treatment with the compound is halted, e.g., aworsening of one or more symptoms of a synucleinopathy. When the secondpre-selected result is observed, administration of the compound to thesubject can be reinstated or increased, or administration of the firsttherapy is reinstated, or the subject is administered both a compoundand first therapy, or an increased amount of the compound and the firsttherapeutic regimen.

The compound can also be administered with a treatment for one or moresymptoms (e.g., behavioral symptoms) of a synucleinopathy. For example,the compound can be co-administered (e.g., at the same time or by anycombination regimen described above) with, e.g., an anti-depressant or amedicament for treating insomnia, agitation, or anxiety.

Identification of Compounds that Modulate Phosphatase Activity,Geranylgeranyltransferase Activity, ATPase Activity, Ubiquitin-SpecificProtease Activity, or Kinase Activity

Assays for measuring phosphatase (e.g., PPZ2, PTP2, PTC4 of a homologthereof) activity are described in, e.g., Ruiz et al. (2004) J. Biol.Chem. 279(33):34421-34430 (PPZ2); Mattison et al. (2000) Genes & Dev.14:1229-1235 (PTP2); Keen et al. (2005) J. Biol. Chem. 280(33):29519 andMitsuhashi et al. (2005) Mol. Cell Biochem. 269(1-2):183. Thesephosphatase activity assays can be used to identify compounds thatinhibit or enhance the activity of a phosphatase (e.g., PPZ2, PTP2, PTC4or a homolog thereof) described herein. Examples of phosphataseinhibitors, include but are not limited to, okadaic acid, calculin A,tautomycin, microcystin-LR, Fostriecin, Canthardin,Thryrsiferyl-23-acetate, cyclopentaquinoline3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinaline-4,8-dicarboxylic acid,and 3-benzoyl-naptho[1,2-b]furan-4,5-dione (McCluskey et al. (2001) MiniRev. Med. Chem. 1:43-55; Brissen et al. (2004) Mol. Pharm.66(4):824-833).

Assays for measuring geranylgeranyltransferase activity are describedin, e.g., Farnsworth et al. (1994) Proc. Natl. Acad. Sci. USA91(25):11963-11967 and Peterson et al. J. Biol. Chem.281(18):12445-12450. Examples of geranylgeranyltransferase inhibitors,e.g., useful as positive controls for the assays, includeimidazolyl-beta-amino acid derivatives and L-778,123 and are describedin Reid et al. (2004) Biochemistry 43(28):9000-9008; Lin et al. (2004)Bioorg. Med. Chem. Lett. 14(20):5057-5062; and Saha et al. (2005)Bioorg. Med. Chem. Lett. 15(6):1713-1719. These assays can be used toidentify compounds that inhibit the activity of thegeranylgeranyltransferase BET4 described herein.

Assays for measuring ATPase activity are described in, e.g., Tjian etal. (1979) Proc. Natl. Acad. Sci. USA 76(2):610-614; Collins et al.(1986) Proc. Natl. Acad. Sci. USA 83:4799-4803; and Mesngon et al.(2006) J Neurosci. 26(7):2132-2139. Examples ofgeranylgeranyltransferase inhibitors, e.g., useful as positive controlsfor the assays, include omeprazole, geldanamycin, radicicol,cyclopiazonic acid, and lansoprazole and are described in Shinono et al.(2002) Anticancer Res. 22(5):2907-2911. These assays can be used toidentify compounds that inhibit the activity of thegeranylgeranyltransferase BET4 described herein. Assays for measuringubiquitin-specific protease activity are described in, e.g., Holowaty etal. (2003) J Biol. Chem. 278(48):47753-47761; Ovaa et al. (2004) Proc.Natl. Acad. Sci. USA 101:2253-2258; Gilchrist et al. (2000) BiochimBiophys Acta 1481(2):297-309; Angelats et al. (2003) Mamm. Genome14(1):31-46; and Lee et al. (2003) Reproduction, Fertility, andDevelopment 15(2):129-133. These assays can be used to identifycompounds that inhibit the activity of the ubiquitin-specific proteasesdescribed herein.

Assays for measuring kinase activity are described in, e.g., Teige etal. (2001) Proc. Natl. Acad. Sci. USA 98(10):5652-5630 (RCK2); Sedgwicket al. (2006) Biochem. J. 399(1):151-160 (IME2); and Tipper et al.(1983) Arch. Biochem. Biophys. 227(2):386-396 (YSK3, a casein kinase Iisoform). These assays can be used to, e.g., identify compounds thatenhance the activity of a protein kinase such as any of those describedherein.

The following are examples of the practice of the invention. They arenot to be construed as limiting the scope of the invention in any way.

EXAMPLES Example 1 Materials and Methods

Characteristics of a Less-toxic Alpha-Synuclein Strain Used in PlasmidOverexpression Screen

The two copy alpha-synuclein expressing yeast strain used for themodifier screen consisted of aSyn-YFP integrated at the HIS3 and TRP1loci as well as a CEN-based extrachromosomal plasmid with agalactose-inducible promoter to express each putative modifier gene. Thepresence of the extra galactose-inducible promoter (three total) as wellas the different aSyn-YFP integration sites resulted in slightly lesstoxicity compared to the original two copy strain (Outeiro et al. (2003)Science 302:1772). The slightly less toxic strain is referred to asITox2C and the higher toxicity strain is referred to as HTox2C.

Growth Rates and Survivorship Assays

Growth curves were generated by growing cells overnight in syntheticmedia containing 2% raffinose at 30° C. to log phase and then dilutingthem to 0.1-0.3 OD_(600nm.) 2% galactose was then added and OD_(600nm)readings were taken at the indicated time points. Survivorship assayswere performed as described (Haynes et al. (2004) Mol Cell 15:767).Briefly, survivorship was determined by growing strains overnight insynthetic media containing raffinose to log phase followed by theaddition of 2% galactose to induce expression of alpha-synuclein. Tomaintain wild-type cells in log phase several dilutions were madethroughout the time course. At the described time points, 1 OD_(600nm)was harvested, diluted 1:1000 and 300 μl of these cells were plated onsynthetic media containing 2% glucose and incubated at 30° C. Colonyforming units were then determined.

Alpha-Synuclein Toxicity Modifier Screen

4,954 full-length yeast ORFs were amplified by polymerase chain reactionand captured by recombination cloning into a Gateway™ pDONR221 vector(Invitrogen). The clones were sequenced from N-terminus to C-terminusand verified to be wild type. For the expression screen, the clones weretransferred into a galactose-inducible expression plasmid (pBY011; CEN,URA+, ampR) using the Gateway™ technology (Invitrogen). Additionalinformation about the Yeast FLEXGene collection is available atwww.hip.harvard.edu/research/yeast_flexgene/. Plasmid DNA from theexpression clones were isolated using the REAL™ miniprep kit (Qiagen).DNA was dried in individual wells of 96-well microtiter plates andtransformed into a strain expressing alpha-synuclein integrated at theHIS3 and TRP1 locus. A standard lithium acetate transformation protocolwas modified for automation and used by employing a BIOROBOT Rapidplate96-well pipettor (Qiagen). The transformants were grown in syntheticdeficient media lacking uracil (SD-Ura) with glucose overnight. Theovernight cultures were inoculated into fresh SD-Ura media withraffinose and allowed to reach stationary phase. The cells were spottedon to SD-Ura+glucose and SD-Ura+galactose agar plates. Suppressors ofalpha-synuclein induced toxicity were identified on galactose platesafter 2-3 days of growth at 30° C. The screens were repeated threeindependent times and candidate modifier genes were retested at leasttwice to confirm their authenticity. To exclude the possibility of falsepositive toxicity suppressor genes caused by a simple reduction inalpha-synuclein expression, the amount of alpha-synuclein protein wasmonitored by western blotting and flow cytometry. To excludefalse-positive enhancer genes caused by a general inhibition of growthunrelated to alpha-synuclein expression, these genes were transformedinto wild type yeast cells and their effect on growth determined.

Example 2 Plasmid Overexpression Screen Identifies Modifiers ofAlpha-Synuclein Toxicity

A genetic approach was employed to identify critical lethal lesions. Anover-expression library was used in which individual yeast open readingframes were fully sequenced and placed without protein tags under thecontrol of a galactose-inducible promoter. The 4,954 randomly selectedgenes in this library, representing all functional classes, wereindividually transformed into a yeast strain expressing aSyn-WT. A yeaststrain was used that exhibited a slightly lower level of alpha-synucleinexpression than described previously (Outeiro et al. (2003) Science302:1772 and Example 1). The extended time course for toxicity producedsome growth on galactose-containing agar plates, allowing for screeningsimultaneously for enhancers and suppressors of toxicity. Genes wereidentified that either suppressed or enhanced alpha-synuclein toxicitywhen overexpressed (Table 2). One functional class enriched in thescreen provided proof-of-principle for the effectiveness of the screen.These genes were either involved in carbohydrate metabolism andgalactose-regulated gene expression specifically, or produced a moregeneral inhibition of gene expression.

TABLE 2 Yeast Genes that Modulate Alpha-Synuclein Toxicity WhenOverexpressed Gene Name Suppressor or Type of Gene (SGD Gene ID)Enhancer Potential Function Amino Acid DIP5 Suppressor Dicarboxylicamino acid permease, Transport (YPL265W) mediates high-affinity andhigh- capacity transport of L-glutamate and L-aspartate; also atransporter for Gln, Asn, Ser, Ala, and Gly. Carbohydrate MIG1Suppressor Transcription factor involved in Metabolism (YGL035C) glucoserepression; C2H2 zinc finger protein similar to mammalian Egr and Wilmstumor proteins. MIG3 Suppressor Probable transcriptional repressor(YER028C) involved in response to toxic agents such as hydroxyurea thatinhibit ribonucleotide reductase; phosphorylation by Snf1p or the Mec1ppathway inactivates Mig3p, allowing induction of damage response genes.YOR062C Suppressor Protein of unknown function; (YOR062C) similar toYKR075Cp and Reg1p; expression regulated by glucose and Rgt1p. UGP1Suppressor UDP-glucose pyrophosphorylase (YKL035W) (UGPase), catalysesthe reversible formation of UDP-Glc from glucose 1-phosphate and UTP,involved in a wide variety of metabolic pathways, expression modulatedby Pho85p through Pho4p. REG1 Suppressor Regulatory subunit of type 1protein (YDR028C) phosphatase Glc7p, involved in Negative regulation ofglucose- repressible genes. MIG2 Suppressor Protein containing zincfingers, (YGL209W) involved in repression, along with Mig1p, of SUC2(invertase) expression by high levels of glucose; binds to Mig1p-bindingsites in SUC2 promoter. GIS3 Suppressor Protein of unknown function.(YLR094C) multicopy suppressor of the Gal- phenotype of snf1 mig1srb8/10/11 cells SIP5 Enhancer Protein of unknown function; (YMR140W)interacts with both the Reg1p/Glc7p phosphatase and the Snf1p kinase.NRG2 Enhancer Transcriptional repressor that (YBR066C) mediates glucoserepression and negatively regulates filamentous growth; has similarityto Nrg1p. NRG1 Enhancer Transcriptional repressor that (YDR043C)recruits the Cyc8p-Tup1p complex to promoters; mediates glucoserepression and negatively regulates a variety of processes includingfilamentous growth and alkaline pH response. ER To Golgi SEC21Suppressor Gamma subunit of coatomer, a Transport (YNL287W) heptamericprotein complex that together with Arf1p forms the COPI coat; involvedin ER to Golgi transport of selective cargo. SEC28 SuppressorEpsilon-COP subunit of the (YIL076W) coatomer; regulates retrogradeGolgi-to-ER protein traffic; stabilizes Cop1p, the alpha-COP and thecoatomer complex; non- essential for cell growth. SFT1 SuppressorIntra-Golgi v-SNARE, required for (YKL006C-A) transport of proteinsbetween an early and a later Golgi compartment. GLO3 EnhancerADP-ribosylation factor GTPase (YER122C) activating protein (ARF GAP),involved in ER-Golgi transport; shares functional similarity with Gcs1p.TRS120 Enhancer One of 10 subunits of the transport (YDR407C) proteinparticle (TRAPP) complex of the cis-Golgi which mediates vesicle dockingand fusion; involved in endoplasmic reticulum (ER) to Golgi membranetraffic. YIP3 Enhancer Protein localized to COPII vesicles, (YNL044W)proposed to be involved in ER to Golgi transport; interacts with membersof the Rab GTPase family and Yip1p. BET4 Enhancer Alpha subunit of TypeII (YJL031C) geranylgeranyltransferase required for vesicular transportbetween the endoplasmic reticulum and the Golgi; provides a membraneattachment moiety to Rab-like proteins Ypt1p and Sec4p. SLY41 EnhancerProtein involved in ER-to-Golgi (YOR307C) transport. GOS1 Enhancerv-SNARE protein involved in Golgi (YHL031C) transport, homolog of themammalian protein GOS-28/GS28 SEC31 Enhancer Essential phosphoprotein(YDL195W) component (p150) of the COPII coat of secretory pathwayvesicles, in complex with Sec13p; required for ER Kinase RCK2 SuppressorProtein kinase involved in the (YLR248W) response to oxidative andosmotic stress; identified as suppressor of S. pombe cell cyclecheckpoint mutations. IME2 Suppressor Serine/threonine protein kinase(YJL106W) involved in activation of meiosis, associates with Ime1p andmediates its stability, activates Ndt80p; IME2 expression is positivelyregulated by Ime1p. YCK3 Suppressor Palmitoylated, vacuolar membrane-(YER123W) localized casein kinase I isoform; negatively regulatesvacuole fusion during hypertonic stress via phosphorylation of the HOPScomplex subunit, Vps41p; shares overlapping essential functions withHrr25p. KSP1 Suppressor Nonessential putative (YHR082C) serine/threonineprotein kinase of unknown cellular role; overproduction causes allele-specific suppression of the prp20-10 mutation. Phosphatase PTP2Suppressor Phosphotyrosine-specific protein (YOR208W) phosphataseinvolved in the inactivation of mitogen-activated protein kinase (MAPK)during osmolarity sensing; dephosporylates Hog1p MAPK and regulates itslocalization; localized to the nucleus. PTC4 Suppressor Cytoplasmic type2C protein (YBR125C) phosphatase; identified as a high- copy numbersuppressor of the synthetic lethality of a cnb1 mpk1 double deletion;overexpression decreases high-osmolarity induced Hog1p phosphorylationand kinase activity. PPZ2 Enhancer Serine/threonine protein (YDR436W)phosphatase Z, isoform of Ppz1p; involved in regulation of potassiumtransport, which affects osmotic stability, cell cycle progression, andhalotolerance. TOR Pathway LST8 Suppressor Protein required for thetransport of (YNL006W) amino acid permease Gap1p from the Golgi to thecell surface; component of the TOR signaling pathway; associates withboth Tor1p and Tor2p; contains a WD-repeat. Transcription/ ZDS2Suppressor Protein that interacts with silencing Translation (YML109W)proteins at the telomere, involved in transcriptional silencing; paralogof Zds1p. ZDS1 Suppressor Protein that interacts with silencing(YMR273C) proteins at the telomere, involved in transcriptionalsilencing; has a role in localization of Bcy1p, a regulatory subunit ofprotein kinase A; implicated in mRNA nuclear export. SKO1 SuppressorBasic leucine zipper (bZIP) (YNL167C) transcription factor of theATF/CREB family that forms a complex with Tup1p and Ssn6p to bothactivate and repress transcription; cytosolic and nuclear proteininvolved in the osmotic and oxidative stress responses. HAP4 SuppressorSubunit of the heme-activated, (YKL109W) glucose-repressedHap2p/3p/4p/5p CCAAT-binding complex, a Transcriptional activator andglobal regulator of respiratory gene expression; provides the principalactivation function of the complex. VHR1 Suppressor Transcription factor(YIL056W) TIF4632 Suppressor Translation initiation factor eIF4G,(YGL049C) subunit of the mRNA cap-binding protein complex (eIF4F) thatalso contains eIF4E (Cdc33p); associates with the poly(A)-bindingprotein Pab1p, also interacts with eIF4A (Tif1p); homologous toTif4631p. SUM1 Suppressor Transcriptional repressor required (YDR310C)for repression of middle sporulation-specific genes during mitosis;regulated by the pachytene checkpoint; a dominant mutation acts as asuppressor of silencing defects of SIR2 mutations. STB3 SuppressorProtein that binds Sin3p in a two- (YDR169C) hybrid assay. CUP9Suppressor Homeodomain-containing (YPL177C) transcriptional repressor ofPTR2, which encodes a major peptide transporter; imported peptidesactivate ubiquitin-dependent proteolysis, resulting in degradation ofCup9p and de-repression of PTR2 transcription. JSN1 Suppressor Member ofthe Puf family of RNA- (YJR091C) binding proteins, interacts with mRNAsencoding membrane- associated proteins; overexpression suppresses atub2-150 mutation and causes increased sensitivity to benomyl inwild-type cells. MATALPHA1 Enhancer Transcriptional co-activator(YCR040W) involved in regulation of mating- type-specific geneexpression; targets the transcription factor Mcm1p to the promoters ofalpha- specific genes; one of two genes encoded by the MATalpha matingtype cassette. MKS1 Enhancer Pleiotropic negative transcriptional(YNL076W) regulator involved in Ras-CAMP and lysine biosyntheticpathways and nitrogen regulation; involved in retrograde (RTG)mitochondria-to- nucleus signaling Trehalose NTH1 Suppressor Neutraltrehalase, degrades (YDR001C) trehalose; required for thermotoleranceand may mediate resistance to other cellular stresses; may bephosphorylated by Cdc28p. Ubiquitination HRD1 SuppressorUbiquitin-protein ligase required for (YOL013C) endoplasmicreticulum-associated degradation (ERAD) of misfolded proteins;genetically linked to the unfolded protein response (UPR); regulatedthrough association with Hrd3p; contains an H2 ring finger. SAN1Suppressor Ubiquitin-protein ligase, controls (YDR143C) turnover of aspecific class of unstable nuclear proteins including Sir4p but notSir2p or Sir3p; san1 mutations suppress sir4, spt16, and cdc68mutations, suggesting a role in chromatin silencing. UBP7 EnhancerUbiquitin-specific protease that (YIL156W) cleaves ubiquitin-proteinfusions. UBP11 Enhancer Ubiquitin-specific protease that (YKR098C)cleaves ubiquitin from ubiquitinated proteins. Other PFS1 SuppressorSporulation protein required for (YHR185C) prospore membrane formationat selected spindle poles, ensures functionality of all four spindlepole bodies of a cell during meiosis II; not required for meioticrecombination or meiotic chromosome segregation. MUM2 SuppressorCytoplasmic protein essential for (YBR057C) meiotic DNA replication andSporulation; interacts with Orc2p, which is a component of the originrecognition complex. OSH3 Suppressor Member of an oxysterol-binding(YHR073W) protein family with seven members in S. cerevisiae; familymembers have overlapping, redundant functions in sterol metabolism andcollectively perform a function essential for viability. PIN4 SuppressorProtein involved in G2/M phase (YBL051C) progression and response to DNAdamage, interacts with Rad53p; contains an RNA recognition motif, anuclear localization signal, and several SQ/TQ cluster domains;hyperphosphorylated in response to DNA damage. MGA2 Suppressor ERmembrane protein involved, (YIR033W) with its homolog Spt23p, inregulation of OLE1 transcription; inactive ER form dimerizes and onesubunit is then activated by ubiquitin/proteasome-dependent processingfollowed by nuclear targeting. OSH2 Suppressor Member of anoxysterol-binding (YDL019C) protein family with seven members in S.cerevisiae; family members have overlapping, redundant functions insterol metabolism and collectively perform a function essential forviability. URE2 Suppressor Nitrogen catabolite repression (YNL229C)regulator that acts by inhibition of GLN3 transcription in good nitrogensource; altered form of Ure2p creates [URE3] prion. ISN1 SuppressorInosine 5′-monophosphate (IMP)- (YOR155C) specific 5′-nucleotidase,catalyzes the breakdown of IMP to inosine, does not show similarity toknown 5′-nucleotidases from other organisms. LSG1 Suppressor PutativeGTPase involved in 60S (YGL099W) ribosomal subunit biogenesis; requiredfor the release of Nmd3p from 60S subunits in the cytoplasm. IDS2Enhancer Protein involved in modulation of (YJL146W) Ime2p activityduring meiosis, appears to act indirectly to promote Ime2p-mediated latemeiotic functions; found in growing cells and degraded duringsporulation. IZH3 Enhancer Membrane protein involved in zinc (YLR023C)metabolism, member of the four- protein IZH family, expression inducedby zinc deficiency; deletion reduces sensitivity to elevated zinc andshortens lag phase, overexpression reduces Zap1p activity QDR3Suppressor Multidrug transporter of the major (YBR043C) facilitatorsuperfamily, required for resistance to quinidine, barban, cisplatin,and bleomycin TPO4 Enhancer Polyamine transport protein, (YOR273C)recognizes spermine, putrescine, and spermidine; localizes to the plasmamembrane; member of the major facilitator superfamily Unknown YKL088WSuppressor Protein required for cell viability. (YKL088W) Predictedphosphopantothenoylcysteine decarboxylase, may be involved in coenzyme Abiosynthesis; interacts with Sis2p and Vhs3p YOR291W Suppressor Probablecation-transporting (YOR291W) ATPase 2 YML081W Suppressor Hypotheticalprotein. (YML081W) YBR030W Suppressor Hypothetical protein. (YBR030W)YMR111C Suppressor Hypothetical protein, green (YMR111C) fluorescentprotein (GFP)-fusion protein localizes to the nucleus SGD stands forSaccharomyces Genome Database, which is available at yeastgenome.organd/or ensembl.org/Saccharomyces_cerevisiae.

Other Embodiments

It is to be understood that, while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention. Other aspects, advantages, and modifications of the inventionare within the scope of the claims set forth below.

What is claimed is:
 1. A method of identifying a compound that inhibitsalpha synuclein-mediated toxicity, the method comprising: screening toidentify an agent that inhibits expression or activity of GOS1, SEC31,IZH3, MKS1, TPO4, GOSR1, SEC31A, SEC31B, ADIPOR2, ADIPOR1, NRG1, NRG2,BET4,GLO3, SLY41, TRS120, YIP3, IDS2, PPZ2, UBP11, UBP7, SIP5,MATALPHA1, KLF12, KLF5, ZNF323, ZNF718, ZNF705A, RABGGTA, ZNF289,SLC35E1, NIBP, RABAC1, KIAA0999, PPP1CC, PPP1CA, USP21, or USP2;providing a cell expressing an amount or form of alpha synuclein thatreduces viability of the cell, wherein the cell is a yeast cell;contacting the cell with the agent; and measuring cell viability in thepresence of the agent, wherein an increase in cell viability in thepresence of the agent as compared to cell viability in the absence ofthe agent identifies the agent as a compound that inhibits alphasynuclein-mediated toxicity.
 2. The method of claim 1, wherein thescreening is to identify an agent that inhibits expression or activityof NRG1, NRG2, BET4, GLO3, SLY41, TRS120, YIP3, IDS2, PPZ2, UBP11, UBP7,SIP5, MATALPHA1, KLF12, KLF5, ZNF323, ZNF718, ZNF705A, RABGGTA, ZNF289,SLC35E1, NIBP, RABAC1, KIAA0999, PPP1CC, PPP1CA, USP21, or USP2.
 3. Themethod of claim 1, wherein the screening comprises: providing a cellexpressing GOS1, SEC31, IZH3, MKS1, TPO4, GOSR1, SEC31A, SEC31B,ADIPOR2, ADIPOR1, NRG1, NRG2, BET4, GLO3, SLY41, TRS120, YIP3, IDS2,PPZ2, UBP11, UBP7, SIP5, MATALPHA1, KLF12, KLF5, ZNF323, ZNF718,ZNF705A, RABGGTA, ZNF289, SLC35E1, NIBP, RABAC1, KIAA0999, PPP1CC,PPP1CA, USP21, or USP2 protein; contacting the cell with an agent; andmeasuring the expression of the protein in the presence of the agent,wherein a reduction in the expression of the protein in the presence ofthe agent as compared to the expression of the protein in the absence ofthe agent identifies the agent as a compound that inhibits theexpression of the protein.
 4. The method of claim 1, wherein thescreening comprises: providing a cell comprising a reporter constructcomprising (i) a promoter sequence of a gene encoding GOS1, SEC31, IZH3,MKS1, TPO4, GOSR1, SEC31A, SEC31B, ADIPOR2, ADIPOR1, NRG1, NRG2, BET4,GLO3, SLY41, TRS120, YIP3, IDS2, PPZ2, UBP11, UBP7, SIP5, MATALPHA1,KLF12, KLF5, ZNF323, ZNF718, ZNF705A, RABGGTA, ZNF289, SLC35E1, NIBP,RABAC1, KIAA0999, PPP1CC, PPP1CA, USP21, or USP2, and (ii) a nucleotidesequence encoding a reporter protein; contacting the cell with an agent;and measuring the expression of the reporter protein in the presence ofthe agent, wherein a reduction in the expression of the reporter proteinin the presence of the agent as compared to the expression of thereporter protein in the absence of the agent identifies the agent as acompound that inhibits the expression of the protein.
 5. The method ofclaim 1, wherein the screening comprises: providing an GOS1, SEC31,IZH3, MKS1, TPO4, GOSR1, SEC31A, SEC31B, ADIPOR2, ADIPOR1, NRG1, NRG2,BET4, GLO3, SLY41, TRS120, YIP3, IDS2, PPZ2, UBP11, UBP7, SIP5,MATALPHA1, KLF12, KLF5, ZNF323, ZNF718, ZNF705A, RABGGTA, ZNF289,SLC35E1, NIBP, RABAC1, KIAA0999, PPP1CC, PPP1CA, USP21, or USP2 protein;contacting the protein with an agent; and measuring the activity of theprotein in the presence of the agent, wherein a reduction in theactivity of the protein in the presence of the agent as compared to theactivity of the protein in the absence of the agent identifies the agentas a compound that inhibits the activity the protein.